Method of treating pulmonary disease with interferons

ABSTRACT

A method of treating a pulmonary disease such as, for instance idiopathic pulmonary fibrosis (IPF), mixed connective tissue disease and asthma, comprising administering an aerosolized interferon such as interferon γ in a therapeutically effective amount is provided herein. Also, pharmaceutical compositions of one or more aerosolized interferon(s) alone or in combination with other therapeutic agents are provided.

This application is continuation in part of U.S. Ser. No. 12/322,440,filed Feb. 3, 2009 which is a division of U.S. Ser. No. 11/231,322,filed Sep. 20, 2005.

GOVERNMENT SUPPORT

Some research leading to the present invention was supported in part byresearch NIH grant R01HL55791, K07 HL03030, and M01 RR00096. Thegovernment may have certain rights in the present invention.

FIELD OF THE INVENTION

This invention relates to methods of treating pulmonary diseases usingaerosol interferons, formulations of one or more interferons for aerosoldelivery and methods for determining aerosol deposition.

BACKGROUND

The mainstay of asthma treatment according to current NAEPP/NIHguidelines remains anti-inflammatory agents, of which corticosteroidsare the most potent. However, long term administration ofcorticosteroids is associated with systemic side effects. Furthermore,some asthmatics are resistant to corticosteroids. Therefore, there is aneed for new agents aimed at the inflammatory response in allergicairway disease.

The immune mechanism of asthma involves the polarized involvement ofmemory CD4⁺ T-helper cell with an imbalance of cells secreting type 2(Th2) cytokines (interleukin (IL)-4, IL-5). The cytokine interferon-γ(INF-γ) is required for naive CD4⁺ lymphocyte differentiation to Th1phenotype.

Airways inflammation in asthma is characterized by the presence of anincreased number of eosinophils and activated CD4⁺ T cells. Asthmainvolves the polarized involvement of memory CD4⁺ T helper cells with animbalance of cells secreting Th2-type cytokines over those secretingTh1-type cytokines. There is increased production of a number ofcytokines including Type 2 cytokines IL-4 and IL-5, tumor necrosisfactor (TNF)-a, and granulocyte-macrophage colony-stimulating factor(GM-CSF) as well as tissue eosinophilia and increased IgE production.Most studies of cytokine profiles in airway inflammation come from themurine model of asthma. Animals are sensitized and challenged withantigen, usually ovalbumin and are found to have antigen specific IgEproduction, airway eosinophilia and airway hyperresponsiveness toaerosol antigen challenge. These changes are associated with increasedTh2 cytokines and decreased INF-γ production (Brusselle et al., Am JRespir Cell Mol Biol, 1995 March; 12(3):254-259).

The Th2 cytokine IL-4 plays a prominent role in airway inflammation bypromoting isotype switching of B cells to IgE synthesis and inducingnaive T cell differentiation to Th2 lymphocytes. IL-4 knockout micechallenged with aerosolized antigen failed to produce specific IgE,airway hyperresponsiveness, airway eosinophilia, or Th2 cytokines in theairways (Brusselle et al., Am J Respir Cell Mol Biol, 1995 March;12(3):254-259.) Wild-type mice treated with anti-IL-4 during the initialexposure to antigen but not during challenge inhibited IL-5 productionand airways eosinophilia, whereas anti-IL-4 given during antigenchallenge did not inhibit airways eosinophilia, indicating that IL-4 isessential for the induction of a local Th2 response (Coyle et al., Am JRespir Cell Mol Biol 1995 July; 13(1):54-59).

IL-10 is a cytokine produced by Th1 and Th2 lymphocytes, monocytes andmacrophages, mast cells, keratinocytes, and eosinophils. IL-10 acts asan anti-inflammatory cytokine by downregulating the synthesis ofproinflammatory cytokines by different cells, particularly monocyticcells. IL-10 downregulates the production of IL-5 by functionallyinhibiting antigen presenting cells (APC) (Pretolani et al., Res Immunol1997 January). A direct effect of IL-10 on eosinophil function has beendemonstrated as well. Low concentrations of IL-10 were almost as activeas corticosteroids in decreasing CD4 expression on eosinophils andaccelerating cell death. GM-CSF is a cytokine directly involved in thehoming and activation of eosinophils and neutrophils in inflamedtissues. Diminished levels of IL-10 production by PBMC and alveolarmacrophages have been noted in asthmatic patients compared to normalcontrols (Borish, L et al., J Allergy Clin Immunol 1996 June;97(6):1288-1296; Koning et al., Cytokine 1997 June; 9(6):427-436). Intwo models of allergic inflammation in mice, instillation of IL-10protected sensitized mice from airway eosinophilia and neutrophiliapossibly by inhibiting IL-5 and TNF-a (Zuany-Amorim et al., J ClinInvest 1996:2644-2651; Zuany-Amorim et al., J Immunol 1996 Jul. 1;157(1):377-84).

Consistent with the Th2/Th1 dichotomy of cytokine production, murinemodels of asthma observe a cytokine profile of IL-4 and IL-5predominance and low levels of the Th1 cytokines INF-γ and IL-12(Ohkawara et al., Am J Respir Cell Mol Biol 1997 May; 16(5):510-20).Recent animal studies look at treatment with recombinant murine IL-12 inan attempt to reverse Th2 predominance. In vitro data indicate that thepresence of IL-12 during the primary antigen stimulation ofT-lymphocytes favors the development of Th1 cells (Kips et al., Am JRespir Crit Care Med 1996 February; 153(2):535-9). Kips confirmed thisin vivo by administering IL-12 at the time of immunization andpreventing production of specific IgE, airway eosinophilia, and airwayhyperreactivity. Although, IL-12 administration during the aerosolchallenge of already sensitized mice prevented airway eosinophilia andairway hyperresponsiveness, it did not decrease specific IgE production,suggesting that IL-12 stimulates the differentiation of naive Th cellsinto Th1 cells, and can suppress the development of Th2 cells.Inhibition of antigen induced airway eosinophilia by IL-12 is INF-γdependent during the initial sensitization, but becomes INF-γindependent during the secondary challenge (Brusselle et al., Am JRespir Cell Mol Biol 1997 December; 17(6):767-71). In addition, mucosalgene transfer of IL-12 gene in the lung via vaccinia virus vector tosensitized mice prior to aeroallergen challenge has been demonstrated tolead to suppression of IL-4, IL-5, airway hyperresponsiveness, andairway eosinophilia in an INF-γ dependent manner (Hogan et al.,—Eur JImmunol 1998 February; 28(2):413-23).

Increasing INF-γ levels may drive the immune response to a Th1 phenotypeand may be beneficial in asthma. Clinical correlation in humans hasfocused on cytokine levels in serum or stimulated PBMC. Mostmeasurements of cytokines using stimulated PBMC have been performed inchildren. These studies have demonstrated an increased propensitytowards IL-4 and IL-5 production and decreased production of INF-γ isasthmatic children. Furthermore, others have demonstrated an inverseassociation between atopy and/or asthma severity and release of INF-γ(Imada et al., (1995) Immunology 85(3): 373-80; Corrigan et al., (1990)Am Rev Respir Dis 141(4) Pt 1: 970-7; Leonard et al., (1997) Am J RespirCell Mol Biol 17(3): 368-75; Kang et al., (1997) J Interferon CytokineRes 17(8): 481-7). Cytokine levels in BAL fluid from asthmatic patientsreveal low levels of INF-γ (Kang et al., (1997) J Interferon CytokineRes 17(8): 481-7).

Clinical trials of rINF-γ in humans are few. As of 1999, INF-γ isindicated for the treatment of chronic granulomatous disease in whichprolonged treatment (average duration 2.5 years) was associated withimprovement in skin lesions, with minimal adverse events (fever,diarrhea, and flu-like illness) (N Engl J Med 324 (8):509-16; Bemilleret al. (1995) Blood Cells Mol Dis 21(3): 239-47; Weening et al., (1995)Eur J Pediatr 154(4): 295-8). Boguniewicz treated 5 patients with mildatopic asthma with escalating doses of aerosolized r INF-γ (maximum doseof 500 mcg, total study dose of 2400 mcg) delivered over 20 days(Boguniewicz et al., (1995) J Allergy Clin Immunol 95(1) Pt 1: 133-5).All patients tolerated the nebulized r INF-γ but there were nosignificant changes in the endpoints evaluated which included peak flow.

Nebulized r INF-γ was administered to 5 patients with persistent acidfast bacilli (AFB) smear and culture positive multiple-drug resistanttuberculosis (TB) (Condos et al., (1997) Lancet 349(9064): 1513-5).Patients received aerosol r INF-γ, 500 mcg, 3 times weekly for 4 weeks(total study dose 6000 mcg). Therapy was tolerated well with minimalside effects. At the end of the 4 weeks, 4 of the 5 patients were sputumAFB-smear negative and the time to positive culture increased indicatinga reduced organism load after treatment. Interestingly, in thesereported and in additional patients, PEFR performed 1 hour aftertreatment improved by 6% (n=10).

The idiopathic interstitial pneumonias have been grouped into sevencategories based upon histology. They include usual interstitialpneumonia (UIP), non-specific interstitial pneumonia (NSIP), diffusealveolar damage (DAD), organizing pneumonia (OP), desquamativeinterstitial pneumonia (DIP), respiratory bronchiolitis (RB), andlymphocytic interstitial pneumonia (LIP). See, e.g. Nicholson,Histopathology, 2002, 41, 381-391; White, J Pathol 2003, 201, 343-354.

The term “idiopathic pulmonary fibrosis” (IPF), synonymous with“cryptogenic fibrosing alveolitis” (CFA) is the clinical term for amajor subgroup of the idiopathic interstitial pneumonias, and itdescribes a disease characterized by idiopathic progressive interstitialdisease with a mean survival from the onset of dyspnea of 3 to 6 years.A diagnosis of idiopathic pulmonary fibrosis is made by identifyingusual interstitial pneumonia (UIP) on lung biopsy. The histologicalpattern is characterized by heterogeneity that includes patchy chronicinflammation (alveolitis), progressive injury (small aggregates ofproliferating myofibroblasts and fibroblasts, termed fibroblastic foci)and fibrosis (dense collagen and honeycomb change). (See, e.g. King etal., 2000, Am J of Resp. and Critical Care Med., 164, 1025-1032).Treatment of another subgroup of interstitial pneumonia is notpredictive of successful therapy for idiopathic interstitial fibrosis.

Corticosteroids and cytotoxic agents have been a mainstay of therapy,with only 10-30% of patients showing an initial transient response,suggesting the need for long-term therapy (Mapel et al. (1996) Chest110:1058-1067; Raghu et al. (1991) Am. Rev. Respir. Dis. 144:291-296).Due to the poor prognosis of patients with idiopathic pulmonaryfibrosis, new therapeutic approaches are needed.

Interferons are a family of naturally-occurring proteins that areproduced by cells of the immune system. Three classes of interferonshave been identified, alpha, beta and gamma. Each class has differenteffects though their activities overlap. Together, the interferonsdirect the immune system's attack on viruses, bacteria, tumors and otherforeign substances that may invade the body. Once interferons havedetected and attacked a foreign substance, they alter it by slowing,blocking, or changing its growth or function.

Interferon-γ is a pleiotropic cytokine that has specificimmune-modulating effects, e.g. activation of macrophages, enhancedrelease of oxygen radicals, microbial killing, enhanced expression ofMHC Class II molecules, anti-viral effects, induction of the induciblenitric oxide synthase gene and release of NO, chemotactic factors torecruit and activate immune effector cells, down regulation oftransferrin receptors limiting microbial access to iron necessary forsurvival of intracellular pathogens, etc. Genetically engineered micethat lack interferon-γ or its receptor are extremely susceptible tomycobacterial infection.

Recombinant INF-γ was administered to normal volunteers and cancerpatients in the 1980s through intramuscular and subcutaneous routes.There was evidence of monocyte activation, e.g. release of oxidants.Jaffe et al. reported rINFγ administration to 20 normal volunteers.(See, Jaffe et al., J Clin Invest. 88, 297-302 (1991)) First, they gaverINF-γ 250 μg subcutaneously noting peak serum levels at 4 hours and atrough at 24 hours.

Several clinical trials were sponsored to evaluate INF-γ for infectiousdiseases. The MDR-TB clinical trial, entitled “A Phase II/III Study ofthe Safety and Efficacy of Inhaled Aerosolized Recombinant Interferon-γ1 b in Patients with Pulmonary Multiple Drug Resistant Tuberculosis(MDR-TB) Who have Failed an Appropriate Three Month Treatment,” enrolled80 MDR-TB patients at several sites (Cape Town, Port Elizabeth, Durban,Mexico) and randomized them to receive aerosol rINF-γ (500 μg MWF) orplacebo for at least 6 months in addition to second line therapy. Thisclinical trial was stopped prematurely due to lack of efficacy on sputumsmears, M tb culture, or chest radiograph changes.

Ziesche et al. gave rINF-γ subcutaneously at a dose of 200 mg threetimes a week in addition to oral prednisone to 9/18 patients withidiopathic pulmonary fibrosis (IPF). See, Ziesche et al., (1999) N. Eng.J. Med., 341, 1264-1269). The results of a subsequent phase 3 clinicaltrial of interferon γ-1b therapy for IPF were recently published.Although this was the first clinical trial of IPF that had an adequatesample size and was a randomized, prospective, double-blind,placebo-controlled study, no significant effect on markers ofphysiologic function, such as forced vital capacity, was observed.However, more deaths occurred in the placebo group, and survival wassignificantly better for a subset of patients who received interferonγ-1b therapy and had a forced vital capacity of 55% or greater anddiffuse lung capacity for carbon monoxide of 35% or greater of thenormal predicted values. The discordance between disease progression andsurvival in that study remains to be explained. One possibility is thatinterferon γ-1b therapy improves host defense against infection anddiminishes the severity of lower respiratory tract infection when itcomplicates the clinical course of patients with IPF. This possibilityis supported by the observation by Strieter et al. that theinterferon-inducible CXC chemokine, I-TAC/CXCL11, which hasantimicrobial properties, was significantly up-regulated in plasma andbronchoalveolar lavage (BAL) fluid in individuals who receivedinterferon γ-1b compared to those who received placebo, whereasprofibrogenic cytokines were generally not significantly altered byinterferon γ-1b therapy over a 6-month treatment period. (See, Strieteret al., Am J Respir Crit Care Med. (2004). One possibility to explainthe lackluster results is inadequate levels of drug delivered to thelung interstitium with current dosing strategies.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention features a method of treating apulmonary disease in a subject suffering from a pulmonary disease,comprising administering an aerosolized interferon in a therapeuticallyeffective amount. In many embodiments, the pulmonary disease is anobstructive pulmonary disease. In some embodiments the pulmonary diseaseis asthma or idiopathic pulmonary fibrosis. In one embodiment, theimproved symptoms of the pulmonary disease may be measured by anincrease of at least about 1, 2, 3, 4, 5, 7, or 10% or more of predictedforced vital capacity (FVC) relative to values measured in patientsreceiving treatment with a placebo over a period of at about six,twelve, eighteen months, or two or three years or more, preferably atleast about a 12% or at least about 15% or even 20% increase in FVC. Inanother embodiment, the treatment results in a reduction in mortalityamong patients receiving treatment of at least 5%, 10%, 25%, 50% or moreover a period of about two or three years or more. The interferon may beinterferon α, interferon β or interferon γ.

In another embodiment, the subject suffering from the pulmonary disease,such as, for instance, IPF, chronic obstructive pulmonary disease (COPD)or asthma, is unresponsive to treatment with one or more of acorticosteroid, cyclophosphamide, and azathioprine. Furthermore, inpatients that are minimally responsive to immunosuppressant therapies,wherein there is a modest, but insignificant improvement in pulmonaryfunction tests, it is a further aspect of the invention to combinetreatment of these patients with an aerosolized interferon whilemaintaining treatment with one or more other therapeutic regimens,including but not limited to treatment with one or moreimmunosuppressive or anti-inflammatory agents.

In more specific embodiments, aerosolized interferon is administered indoses ranging from 10 μg to 1000 μg, preferably about 50 μg to 750 μg or75 μg to 500 μg or 100 μg to 250 μg, preferably given in a nebulizerfrom one to ten times per week, preferably about two, three, four orfive times per week. In another embodiment, a dose of 100 μg to 500 μgis given in a nebulizer three times per week. Lower doses may be givendepending on the efficiency of the nebulizer. When it is desired totreat patients with a combination of interferon-γ therapy and othertreatment modalities, the aerosolized interferon-γ may be titrated toensure no undesirable effects are experienced by these patients.Furthermore, when combination therapy is a consideration, the otheragents may be delivered by a means in which they are considered to bethe most effective. This may include intravenous, intramuscular,subcutaneous, or may be combined with INF-γ and delivered as an aerosol.

In still other specific embodiments aerosolized interferon isadministered in doses and for time periods and by devices such asnebulizers sufficient to provide INF-γ that may be measured in thebronchoalveolar lavage fluid (BAL) of patients. The INF-γ may be presentin the BAL fluid in amounts of at least 10, 25, 50, 100, 150, 200, 250,300, 500 or even 750 or more picograms/milliliter. Further, in otherspecific embodiments aerosolized interferon is administered in doses andfor time periods sufficient to produce a measurable decrease in thelevel of IL-8 present in the bronchoalveolar lavage (BAL) fluid of apatient of for instance, 10%, 20%, 30%, 40%, 50% or more. In someinstances, the level of IL-8 in the BAL of a patient suffering from apulmonary disease may be reduced to an amount that is no more than 100%,50%, 25%, or 10% more than the level of IL-8 in the BAL fluid of anormal control substantially free of a pulmonary disease. In yetadditional specific embodiments aerosolized interferon is administeredin doses and for time periods sufficient to produce a measurableincrease in the level of TGF-β present in the bronchoalveolar lavage(BAL) fluid of a patient of from 5%, 10%, 15%, 20%, 25%, or even 50%,60%, 75% or 100% or more. The TGF-β may be measured in the BAL fluidafter treatment in some instances in amounts of about 0.25, 0.50, 0.75,1.00, 1.25, 1.50, 1.75, or 2.00 picograms/milliliter or more. In manyinstances, the aerosolized interferon is administered preferably in anebulizer from one to ten times per week, preferably about two, three,four or five times per week. In each of these instances the interferonmay be administered for time periods of one week, two weeks, five weeks,ten weeks, twenty weeks, thirty weeks, forty weeks, 50 weeks, sixtyweeks, seventy weeks or more to attain the desired IL-8, TGF-β, or INF-γlevels.

In some embodiments, the nebulizer is chosen so as to provide deliveryof at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% or even75% or more of the interferon present in the nebulizer to the lung, someprovided to the middle lobe of the lung. It is desirable that no morethan about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% of theinterferon administered be deposited in the gastrointestinal tract, suchas, for instance, in the stomach. Moreover, it is desirable that no morethan about 5%, 10%, 15%, 20%, 25%, 50% or 75% of the interferon remainundelivered to a patient in a drug delivery container of a nebulizer.Still further, it is desirable that no more than about 5%, 10%, 15%,25%, 30%, 35%, 40%, or 50% of the interferon remain in the oropharynx ormouth of a patient.

In another aspect, the invention features a method of accuratelydetermining upper respiratory airway deposition of an agent administeredby aerosol delivery. In one embodiment of this aspect of the invention,the agent administered via aerosol delivery is an interferon such asinterferon α, interferon β or interferon-γ. This technology is uniqueand applies to the delivery of an interferon such as interferon α,interferon β or INF-γ to patients with all types of lung disease.

Other objects and advantages will become apparent from a review of theensuing detailed description taken in conjunction with the followingillustrative drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes a typical tidal breathing pattern.

FIG. 2 describes a reduction in inspiratory flow and a greatly prolongedinspiratory time characteristic of a method of slow and deep inspirationas compared to tidal breathing.

FIG. 3 represents a deposition pattern in a human subject inhaling 4.5μm aerosols using the slow and deep breathing pattern. The imagesdemonstrate minimal deposition of aerosol (less than 10%) in the upperairways illustrated by the small amount of activity in the stomach. Thedeposition image represents radiolabeled aerosol deposited in the lungperiphery of a human subject after 3 breaths using the slow and deeppattern with an inspiratory time of approximately 8 seconds.

FIG. 4 is an illustrative scan in the same subject following 20 breathsof tidal breathing of 1.5 μm particles which is the present standardmode of inhalation. Analysis of the images indicates that the slow anddeep method of breathing which incorporates the use of large particles,slow inspiration and a prolonged inspiratory time is 51 times moreefficient per breath in depositing aerosol particles in the lung.

FIG. 5 depicts a deposition scan of a patient suffering with mixedconnective tissue disease who has been treated three times per week fortwelve weeks with 500 μg of INF-γ delivered via a nebulizer. Imaging wasperformed following a treatment. Regions of interest are shown asoutlines. The deposition image is following inhalation of radiolabeledINF-γ aerosol; INF-γ deposited in lung=54 μg; sC/P=1.28, consistent withperipheral deposition. sC/P means the specific central to peripheralratio described below. a/Xe means the aerosol to xenon ratio.

FIG. 6 represents TGF-β levels measured via BAL before and after aerosoltherapy.

FIG. 7 demonstrates the increased percent predicted total lung capacityafter treatment for five patients with IPF. All patients reportedsubjective improvements in their shortness of breath. By the end ofthree months of treatment, patients in the study had a statisticallysignificant increase in total lung capacity.

FIG. 8 demonstrates the increased percent predicted forced vitalcapacity after treatment in three of the five patients treated in astudy of aerosol rINF-γ for five patients with IPF.

FIGS. 9A and 9B demonstrate the reduction of TGF-β (per mg totalprotein) in the five patients treated with aerosol rINF-γ for IPF. TGF-βis one of the key mediators of fibrosis in the lung. Its activationleads to collagen production. Decreases in its levels should lead toless collagen deposition and less fibrosis in the lung.

FIG. 10 demonstrates the amount of interferon-γ measured in the lungs oftuberculosis patients and patients with idiopathic pulmonary fibrosisboth before and after aerosol treatment with interferon-γ.

FIG. 11 represents the percentage change in peak flow in asthma patientsafter treatment with aerosol INF-γ. All patients receiving aerosolinterferon-γ were studied with spirometry to assess reversible airwaysdisease. At each aerosol treatment, patients had monitoring of peakflows before and after treatment.

FIG. 12 provides a summary of the percent change in peak flowmeasurements referred to in FIG. 2. The average peak flow increasedafter aerosol interferon γ, with significant increases in a fewpatients. Of note, in all patients where peak flow measurementsdecreased after interferon γ, none developed cough or other complaints.These data indicate that aerosol interferon γ is safe and well toleratedin patients with airway disease.

FIG. 13 provides the levels of interferon-γ measured in the BAL fluid ofsix patients both before and after treatment with aerosolizedinterferon-γ. Units are picograms of interferon-γ per ml of BAL fluid.

FIG. 14 depicts the levels of IL-8 measured in the BAL fluid of sixpatients both before and after treatment with aerosolized interferon-γ.Units are picograms of IL-8 per ml of BAL fluid.

FIG. 15 demonstrates the change in forced vital capacity (FVC)graphically among ten IPF patients treated with INF-γ compared to threecontrol groups.

FIG. 16 demonstrates the change in FVC graphically among ten IPFpatients treated with INF-γ compared to three control groups over aperiod of 30 weeks, nine IPF patients over a period of 40 weeks, and sixIPF patients over a period of fifty weeks. One group of patientsreceived no treatment at all (NYU Control Group), one group of patientsreceived acetyl cysteine, one group of patients received etanercept, andtwo groups of patients received perfenadone. Three groups receivedplacebo. These results demonstrate that patients suffering from IPFexperience not only arrest of further deterioration but actual improvedpulmonary function as demonstrated by increased FVC over time. To thecontrary, patients receiving acetyl cysteine, etanercept, perfenadone orno treatment whatsoever demonstrate continued deterioration of pulmonaryfunction as evidenced by decreased FVC over time.

FIG. 17 depicts the levels of IL-8 measured in the BAL fluid of sixpatients both before and after treatment with aerosolized interferon-γand the levels of IL-8 measured in the BAL fluid of four normalpatients. Units are picograms of IL-8 per ml of BAL fluid.

FIG. 18 depicts the levels of INF-γ measured in the BAL fluid of sixpatients both before and after treatment with aerosolized interferon-γand the levels of INF-γ measured in the BAL fluid of four normalpatients. The units are picograms of INF-γ per ml of BAL fluid.

DETAILED DESCRIPTION

Before the present methods and treatment methodology are described, itis to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting, since the scope of the presentinvention will be limited only by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and described the methodsand/or materials in connection with which the publications are cited.

DEFINITIONS

The term “improved symptoms,” in a specific embodiment, is assessed asan improvement of at least 1% of predicted FVC relative to values priorto treatment.

The phrase “unresponsive to treatment with one or more ofcorticosteroid, cyclophosphamide, and azathioprine” means a patientpopulation that is unresponsive to conventional prior art treatments.

Forced vital capacity (VC) means the total amount of air that can bemoved in and out of the lungs.

Fev1 means the forced expiratory volume of air in one second.

Fev1/FVC ratio means the ratio of forced expiratory volume in one secondand forced vital capacity.

The term “pulmonary disease” refers to any pathology affecting at leastin part the lungs or respiratory system. The term is meant to encompassboth obstructive and non-obstructive conditions such as, for instance,asthma, emphysema, chronic obstructive pulmonary disease, pneumonia,tuberculosis, mixed connective tissue disease and fibrosis in all itsforms including but not limited to idiopathic pulmonary fibrosis.

The term “obstructive pulmonary disease” refers to any pulmonary diseasethat results in reduction of airflow in or out of the respiratorysystem. The reduction in airflow relative to normal may be measured intotal or over a finite time, for example, by FVC or FEV1.

The term “idiopathic pulmonary fibrosis” (IPF), synonymous with“cryptogenic fibrosing alveolitis” (CFA) is the clinical term for amajor subgroup of the idiopathic interstitial pneumonias, and itdescribes a disease characterized by idiopathic progressive interstitialdisease with a mean survival from the onset of dyspnea of 3 to 6 years.A diagnosis of idiopathic pulmonary fibrosis is made by identifyingusual interstitial pneumonia (UIP) on lung biopsy. The histologicalpattern is characterized by heterogeneity that includes patchy chronicinflammation (alveolitis), progressive injury (small aggregates ofproliferating myofibroblasts and fibroblasts, termed fibroblastic foci)and fibrosis (dense collagen and honeycomb change).

The term “asthma” refers to a common disease that involves inflammation(cellular injury) and narrowing of the airways leading to the lungs.Asthma occurs in children and adults. Childhood asthma may continue intoadolescence and adulthood, but some adults who develop asthma did nothave asthma when they were younger. Millions of people worldwide areaffected by asthma, which has become more common in recent years.

By “slow and deep breathing” is meant any breathing pattern wherein thetime of inspiration is longer than the time of expiration. Such apattern features a duty cycle (time of inspiration/total time of breath)of greater than 0.5. During normal tidal breathing the duty cycle isalways less than or near 0.5. That is, the time of inspiration is alwaysless than the time for expiration. In disease states, the duty cycledecreases in obstructive disease and for restrictive disorders it islikely to be still less than 0.5. “Slow and deep” breathing may featurean I/E ratio, time of inspiration relative to expiration of greater that1, and in some instances the ratio may approach 8 or 9 thereby yieldinga duty cycle of 0.8 or 0.9

Mechanisms of Action of Interferon-γ

Recombinant interferon-γ is commercially available as ACTIMMUNE® fromInterMune, Brisbane, Calif. Signal transduction pathways have beenrecently studied in cultured cells delineating a temporal regulatorypathway for the response to INF-γ (Vilcek et al., (1994) Int ArchAllergy Immunol 104(4): 311-6; Young et al., (1995) J Leukoc Biol 58(4):373-81). The first events take place when added INF-γ binds to theextracellular domain of its receptor, and leads to tyrosinephosphorylation of preexisting signal transducer and activator oftranscription 1 (STAT-1) at the intracellular domain of the receptor.Only tyrosine-phosphorylated STAT-1 is activated, which allows it toform homodimers (or heterodimers) and bind to a specific DNA sequence.

Upon translocating to the nucleus and binding to its cognate regulatoryelement in the promoters of many genes, STAT-1 activates transcription.STAT-1 can work with other preexisting transcription factors that areconstitutively active, and thus transcription of some genes is maximallyinduced without a need for new protein synthesis. Other genes areregulated by STAT-1 together with transcription factors that are newlysynthesized in response to INF-γ. The IRF-1 gene, which also encodes atranscription factor, is also regulated by STAT-1 in response to INF-γ(Pine, R. (1992) J Virol 66(7): 4470-8; Pine et al., (1994) Embo J13(1): 158-67; Pine et al., (1990) Mol Cell Biol 10(6): 2448-57). Itshould be noted that the promoter of the IRF-1 gene also containsbinding sites for nuclear factor kappa B (NF-kB), which mediates tumornecrosis factor alpha (TNF-α)-activated transcription of the IRF-1 gene(Harada et al., (1994) Mol Cell Biol 14(2):1500-9; R. Pine,unpublished).

Once the IRF-1 protein has been synthesized, it activates transcriptionof a temporally downstream set of genes. IRF-1 has-been shown toregulate the INF-γ-induced expression of key genes involved in antigenprocessing and presentation, including TAP-1, LMP-2, and HLA-A and HLA-Bclass I major histocompatibility antigens (Johnson et al., (1994) MolCell Biol 14(2): 1322-32;

White et al., (1996) Immunity 5(4): 365-76).

IRF-1 is phosphorylated, and manipulating the extent of phosphorylationaffects its DNA-binding activity (Pine et al., (1990) Mol Cell Biol10(6): 2448-57;

Nunokawa et al., (1994) Biochem Biophys Res Commun 200(2): 802-7).

However, there is no clear evidence that phosphorylation of IRF-1 isregulated in vivo. STAT-1 activity is dependent on tyrosinephosphorylation and is affected by the extent of serine phosphorylation.However, the abundance of latent STAT-1 is also regulated. Cells treatedovernight with INF-γ have increased levels of STAT-1 protein, though thetyrosine phosphorylation and DNA-binding activity are only slightlygreater than in unstimulated cells (Pine et al., (1994) Embo J 13(1):158-67).

The study of gene expression and its regulation can provide informationon other aspects of the overall immunological state. Specifically,functional effects of cytokine changes can be confirmed by determinationof specific DNA-binding activities. For example, in T cells IL-12 leadsto activation of STAT-4, while IL-4 leads to activation of STAT-6, theoccurrence of Th1 and Th2 responses or a shift from one to the other maybe reflected in the profile of STAT DNA-binding activities detected at aparticular time (Darnell (1996) Recent Prog Horm Res 51:391-403;Ivashkiv, L. B. (1995) Immunity 3(1): 1-4).

Aerosolized Interferon-γ Treatment of IPF

Recently, a small randomized trial of patients with IPF were treatedwith subcutaneous interferon-γ (INF-γ) (Ziesche et al. (1999) N. Engl.J. Med. 341:1264-1269). Analysis of transbronchial biopsy specimensobtained prior to and six months into therapy with INF-γ, demonstratedthat abnormal pretreatment increases in the profibrotic cytokinestransforming growth factor-β (TGF-β) and connective-tissue growth factor(CTGF) were significantly reduced after treatment with INF-γ (Ziesche etal. (1999) supra). Patients treated with prednisolone alone had nochange iii levels of TGF-β and CTGF.

Delivery of Interferons Aerosol Delivery

In a broad aspect of the invention, a method of treating pulmonarydiseases including asthma and idiopathic pulmonary fibrosis (IPF) in asubject suffering from the pulmonary disease, comprising administeringan aerosolized interferon such as interferon-γ in a therapeuticallyeffective amount wherein the symptoms of the pulmonary disease areimproved or ameliorated. The improved symptoms may be an increase of atleast 10% of predicted FVC relative to values prior to treatment. In apreferred embodiment, aerosolized INF-γ may be used for treatingsubjects suffering from asthma or IPF wherein the subjects areunresponsive to treatment with one or more corticosteroid,cyclophosphamide, and azathioprine. Furthermore, the administration ofan aerosolized interferon such as INF-γ is calculated and optimized inpatients with pulmonary fibrosis. Such administration may result inimprovement in pulmonary function tests in patients.

Interferons such as INF-γ may be administered by several differentroutes, including intravenous, intramuscular, subcutaneous, intranasallyand via aerosol. However, when treating a pulmonary process alone,delivery of medication directly to the lung avoids exposure to otherorgan systems. Effective administration of 500 μg INF-γ via aerosolthree times per week for two weeks has been shown by bronchoalveolarlavage (BAL) analysis in normal patients to result in increased levelsof INF-γ post-administration. Likewise, about 500 micrograms ofinterferon-β three times per week and about 0.25 mg of interferon-αthree times per week is thought to be effective.

It is an object of the present invention to deliver the interferon suchas interferon-γ via the pulmonary route of administration. Interferonslike INF-γ are delivered to the lungs of a mammal while inhaling andtraverses across the lung epithelial lining to the blood stream. (Otherreports of this include Adjei et al., PHARMACEUTICAL RESEARCH, VOL. 7,No. 6, pp. 565-569 (1990); Adjei et al., International Journal ofPharmaceutics, 63:135-144 (1990); Braquet et al., Journal ofCardiovascular Pharmacology, Vol. 13, suppl. 5, s. 143-146 (1989);Hubbard et al., Annals of Internal Medicine, Vol. III, No. 3, pp.206-212 (1989); Smith et al., J. Clin. Invest., Vol. 84, pp. 1145-1146(1989); Oswein et al., “Aerosolization of Proteins”, Proceedings ofSymposium on Respiratory Drug Delivery II, Keystone, Colo., March, 1990;and Platz et al., U.S. Pat. No. 5,284,656. Contemplated for use in thepractice of this invention are a wide range of mechanical devicesdesigned for pulmonary delivery of therapeutic products, including butnot limited to nebulizers, metered dose inhalers, and powder inhalers,all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are the Ultravent® nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn nebulizer,manufactured by Marquest Medical Products, Englewood, Colo.; theVentolin® metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler® powder inhaler, manufactured byFisons Corp., Bedford, Mass., MistyNeb®, manufactured by Allegiance,McGraw Park, Ill.; AeroEclipse®, manufactured by Trudell MedicalInternational, Canada, and the I-Neb® manufactured by PhilipsRespironics.

All such devices require the use of formulations suitable for thedispensing of protein. Typically, each formulation is specific to thetype of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, adjuvants and/orcarriers useful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated. Chemically modified protein may also be prepared indifferent formulations depending on the type of chemical modification orthe type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, may typically comprise protein dissolved in water at aconcentration of about 0.1 to 25 mg of biologically active protein permL of solution. The formulation may also include a buffer and a simplesugar (e.g., for protein stabilization and regulation of osmoticpressure). The nebulizer formulation may also contain a surfactant, toreduce or prevent surface induced aggregation of the protein caused byatomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device may generallycomprise a finely divided powder containing the protein suspended in apropellant with the aid of a surfactant. The propellant may be anyconventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device may comprise afinely divided dry powder containing protein and may also include abulking agent, such as lactose, sorbitol, sucrose, or mannitol inamounts which facilitate dispersal of the powder from the device, e.g.,50 to 90% by weight of the formulation. The protein should mostadvantageously be prepared in particulate form with an average particlesize of less than 10 μm (or microns), most preferably 0.5 to 5 μm, formost effective delivery to the distal lung.

It is a goal of aerosol delivery to significantly increase the deliveryof therapeutic agents such as interferons, including INF-γ, to the deeplung in humans. A particularly preferred approach to breathing slow anddeep inspiration may, when compared with standard (tidal breathing),increase deposition efficiency in the lung periphery by a factor of upto about 50 times.

The specific pattern of breathing using a method of slow and deepinspiration as compared to tidal breathing (FIG. 1) describes areduction in inspiratory flow and a greatly prolonged inspiratory time.This pattern is shown in FIG. 2. The slow inspiration allows aerosolparticles to bypass the upper airways thus making them available fordeposition in the lung. The prolonged inspiration allows for suitablesettling of aerosols in the lung periphery. The prolongation of theinspiratory time and the advanced settling promotes “inspiratorydeposition” before remaining particles can be exhaled. It is possibleunder these circumstances to have almost 100% of the inhaled particlesdepositing before exhalation begins. This process can be furtherenhanced by using particles that are relatively large (e.g., about 4.5μm) that ordinarily would deposit in the oropharynx. The prolongedinspiration of slow and deep breathing is particularly suited fordelivery of drugs to the lungs of patients whose peripheral airwaypathology results in reduced deposition of conventional smaller aerosolsas well as promoting avoidance of deposition in the oropharynx. Diseasesof the lung periphery that may be treated by this method include, forexample, idiopathic pulmonary fibrosis and emphysema. Both theseentities result in enlarged airspaces that result in minimal depositionduring tidal breathing.

This technique of inhalation and deposition can enhance the peripheraldelivery of drug with the intent of promoting systemic absorption intothe systemic circulation via the pulmonary capillaries. FIG. 3represents a deposition pattern in a human subject inhaling 4.5 μmaerosols using the slow and deep breathing pattern. The imagesdemonstrate minimal deposition of aerosol (less than 10%) in the upperairways illustrated by the small amount of activity in the stomach. Thedeposition image represents radiolabeled aerosol deposited in the lungperiphery of a human subject after 3 breaths using the slow and deeppattern with an inspiratory time of approximately 8 seconds. FIG. 4 isan illustrative scan in the same subject following 20 breaths of tidalbreathing of 1.5 μm particles which is the present standard mode ofinhalation. Analysis of the images indicates that the slow and deepmethod of breathing which incorporates the use of large particles, slowinspiration and a prolonged inspiratory time is 51 times more efficientper breath in depositing aerosol particles in the lung.

The manufacture of devices capable of performing the slow and deepmaneuver is complex, but prototype devices that perform this functionare being developed and have been utilized (Profile Therapeutics, Inc.28 State Street, Ste. 1100, Boston, Mass. 02109, which is a subsidiaryof Profile Therapeutics which has its main offices in the UK).

Diseases of the lung parenchyma result in geometric changes in the lungperiphery that can minimize the deposition of inhaled particles.Therapeutics delivered directly to the site of disease (the lungperiphery) can be more effective when compared to the same agentdelivered systemically. A method of slow and deep inhalation of aninterferon, such as INF-γ, aerosol is particularly suited to thetreatment of disease in the alveoli of patients with pulmonary fibrosis.

Human deposition studies have indicated that a slow and deep inhalationmethod is about 50 times more efficient than conventional systems ofaerosol delivery. This breathing pattern allows the design of clinicaltrials to test the efficacy of aerosol therapy for pulmonary diseasessuch as obstructive pulmonary diseases, including, for example,idiopathic pulmonary fibrosis or asthma with agents such as interferons,including, for instance, INF-γ, over a wide range of dosing to the lungperiphery utilizing existing formulations of this agent. Quantitiesdeposited in the lung are controlled by the pattern of breathing becausevirtually no aerosol is exhaled.

Nasal Delivery

Nasal delivery of the protein is also contemplated. Nasal deliveryallows the passage of the protein to the blood stream directly afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextran.

Dosages

It is understood that as further studies are conducted, information willemerge regarding appropriate dosage levels for treatment of variousconditions in various patients, and one of ordinary skill in the art,considering the therapeutic context, age and general health of therecipient, will be able to determine proper dosing. Generally, forinjection or infusion, interferon-γ dosage will be between 100 μg ofbiologically active protein (calculating the mass of the protein alone,without chemical modification) to 750 μg (based on the same) given threetimes per week. More preferably, the dosage may be about 500 μg giventhree times per week. Generally, for injection or infusion, interferon-αdosage is generally 250 to 750 micrograms administered one to five timesper week, preferably about 500 micrograms administered three times perweek. In the instance of interferon-β, dosage is generally 0.10 to 1 mgone to three times per week, preferably about 0.25 mg three times perweek. The dosing schedule may vary, depending on the circulationhalf-life of the protein, and the formulation used.

Administration with Other Compounds

It is a further aspect of the present invention that one may administerthe interferon in conjunction with one or more pharmaceuticalcompositions used for treating a pulmonary disease. Also,anti-inflammatory or immunosuppressive agents may be co-administered,eg. cyclophosphamide, azathioprine or corticosteroids. Administrationmay be simultaneous or may be in serriatim.

It has been shown that after subcutaneous administration of 250 μg INF-γfor three days, there was no increase in BAL levels of INF-γ oralteration of alveolar macrophages, while there was upregulation ofperipheral blood monocytes (Jaffe et al. (1991) J. Clin. Invest.88:297-302). In addition, aerosol INF-γ has been used as adjunctivetherapy in patients with pulmonary tuberculosis.

In the studies described below, patients unresponsive to conventionalimmunosuppressive therapy suffering from IPF were treated withaerosolized INF-γ.

The invention may be better understood by reference to the followingexamples, which are intended to be exemplary of the invention and notlimiting thereof.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the therapeutic methods of the invention and compounds andpharmaceutical compositions, and are not intended to limit the scope ofwhat the inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.), but some experimental errors and deviations shouldbe accounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Patient Population

Study subjects were patients suffering from idiopathic pulmonaryfibrosis (IPF) as diagnosed by the American Thoracic Society criteria Aor B (below). The patient population had failed to respond to or was nota candidate for conventional therapy with corticosteroids,cyclophosphamide, and/or azathioprine. The patient population wastreated with aerosolized INF-γ for twelve weeks.

In the setting of a surgical biopsy showing UIP, these three conditionsmust be met:

1. Exclusion of other known causes of interstitial lung disease, such ascertain drug toxicities, environmental exposures, and connective tissuediseases.2. Abnormal pulmonary function studies that include evidence ofrestriction (reduced vital capacity (VC) often with an increasedFev1/FVC ratio) and/or impaired gas exchange (increasedalveolar-arterial gradient for O₂ or decreased diffusion capacity forCO).3. Bibasilar reticular abnormalities with minimal ground glass opacitieson HRCT scans.

In the absence of a surgical lung biopsy, in an immunocompetent adult, apresumed diagnosis of IPF may be made if:

I. All three above criteria are met.II. A transbronchial lung biopsy (TBBx) or bonchoalveolar lavage (BAL)shows no features to support an alternative diagnosis.III. Three of these four minor criteria:

-   -   1. Age>50    -   2. Insidious onset of unexplained dyspnea on exertion    -   3. Duration of illness>three months    -   4. Bibasilar inspiratory crackles.

Improvement is defined as (1) An increase of 10% of predicted FVC frombaseline value compared to FVC obtained prior to steroid therapy. (2) Ifa patient has a greater than 10% increase in FVC from baseline value andthen returns to baseline value despite therapy.

Patients eligible for inclusion into the study are defined as follows:

Patients diagnosed with IPF based on accepted criteria (see above)within 3 years of screening;

Age 20-70;

A failed trial of prednisone with or withoutcyclophosphamide/azathioprine or patients in whom treatment withsteroids or cytotoxic agents are contraindicated;Patient taking 0-15 mg prednisone or the equivalent for 28 days prior tostudy enrollment and willing to remain on the same dose ofcorticosteroid;FVC≧50% and ≦90% of predicted baseline value at screening;PaO₂≧60 mm Hg at rest on room air;Patient able to understand and willing to sign a written informedconsent and willing to comply with all requirements of the studyprotocol includingPatient fits criteria for research bronchoscopy and is willing toundergo procedure;Patient able to have medication administered three times per week atGCRC unit at Bellevue Hospital.

Patients ineligible for inclusion in the study are defined as follows:(1) Patient unwilling or unable to undergo research bronchoscopy; (2)Patient with known asthma or severe COPD; (3) Patient requiring oxygentherapy for maintenance of adequate arterial oxygenation; (4) Patientwith hypersensitivity to study medication or other component medication;(5) Patient with known severe cardiac disease, severe peripheralvascular disease or seizure disorder which may be exacerbated by studydrug administration (contraindications to drug administration as perpackage insert); (6) Pregnant or lactating females. Females ofchild-bearing age will be required to have negative pregnancy test andbe required to use accepted form of birth control (abstinence for studyduration is the preferred method); (7) Evidence of active infectionwithin one week prior to treatment; (8) Any condition, other than IPF,which is likely to result in the death of the patient within one yearfrom study enrollment; (9) Abnormal serum laboratory values including:(a) Liver function above specified limits: total bilirubin>1.5× upperlimits of normal; alanine amino transferase>3× upper limit of normal;alkaline phosphatase>3× upper limit of normal; albumin<3.0 at screening;(b) CBC outside specified limits: WBC<2,500/mm3; hematocrit<30 or >59;platelets<100,000/mm3; (c) Creatinine>1.5× upper limits normal atscreening; (10) Drugs for therapy for pulmonary fibrosis, excludingcorticosteroids, cyclophosphamide, and/or azathioprine, within theprevious six weeks; (11) Prior therapy with any class of interferonmedication; (12) Investigational therapy for any indication within thelast 28 days.

Example 2

Ten patients were recruited from the IPF registry to be enrolled in anopen label pilot study of aerosolized interferon-γ. The ten patients fitthe inclusion and exclusion criteria. Data collected included pastmedical history including height, weight, and vital signs; personalhistory of all medications and complete occupational and smokinghistory, physical exam, EKG, CBC, electrolyte panel, liver enzymes andcoagulation profile, CXR, chest CT, PFT, ABG, an pregnancy test infemales of child bearing age.

Each patient completed a Pulmonary Fibrosis Questionnaire at thebeginning of the study which questioned extensively the tobaccoexposure, environmental exposures, and medication usage throughout thepatient lifetime. Each patient will complete a symptoms questionnairewhich ascertains tolerability of INF-γ and possible side effects.

Baseline bronchoscopy with bronchoalveolar lavage (BAL) was performed toevaluate the levels of certain pro-fibrotic and inflammatory cytokines.The procedure was performed as follows:

Each patient was evaluated for bronchoscopy as per Bellevue HospitalProtocol. Each evaluation includes Hgb, platelets, BUN/CR, coagulationpanel, ABG with PO2≧75 mm Hg, EKG, CXR. Contradictions to bronchoscopyinclude lack of patient cooperation, recent myocardial infarction,malignant arrhythmias, uncorrectable hypoxemia, unstable bronchialasthma, pulmonary hypertension, partial tracheal obstruction or vocalcord paralysis, bleeding diathesis, and uremia. The patients were NPO atleast 8 hours prior to bronchoscopy. An intravenous line was placed,supplemental oxygen was administered, and continuous pulse oximetry andblood pressure monitoring were performed.

The patients were premedicated with 60 mg IM codeine, viscous lidocainewas applied to the nose and lidocaine gargle and nebulizer (topicalanesthetic bronchoscope) were used. During the procedure, midazolamand/or morphine were sometimes administered to cause sedation anddecrease the cough reflex. These medications are routinely used inbronchoscopy. The bronchoscope was passed through the nose and vocalcords, and an endobronchial exam was performed. BAL was then performedby administering 50 ml aliquots of sterile normal saline, for a total of300 ml, and applying gentle suction for maximum return of fluid.

After BAL fluid was obtained from the patient; it was processed in theGCRC core laboratory under standardized protocol used for processing allBAL. BAL fluid was filtered through sterile gauze. A total cell countwith differential was performed in a hemocytometer. Cell viability wasdetermined by the Trypan Blue method. Twenty cytocentrifuge slides wereprepared from each lobe of BAL fluid and frozen at −70° C. 24 hoursupernatants were collected at a concentration of 10⁶ cells/ml forcytokine ELISA assays. The volume of epithelial lining fluid wasdetermined according to the protein method. Following centrifugation,BAL fluid supernatant was concentrated 10×-50×using the AMICON filtermethod. Cytokine assays were carried out with commercially availablekits (R&D Systems, Minneapolis, Minn.). All samples were assayed intriplicate, and the amount of cytokine was quantified at the end of theassay by a microtiter plater reader. Transbronchial biopsy specimenswere processed for isolation of fibroblasts as previously described(Raghu et al. (1989) Am. Rev. Resp. Dis. 140:95-100) and analyzed forcollagen production using ³H proline incorporation into collagenousproteins. Each patient was monitored for potential side effects ofbronchoscopy, including but not limited to fever, shortness of breath,hemoptysis, and pneumothorax for 4 hours post procedure in the GCRC bythe clinical nursing staff. Concomitant medications were recorded in thepatient's medical record.

Investigational therapies are not permitted while the patient is on thestudy. Pre-clinical rat studies have shown that parenteral INF-γdecreases the concentration of hepatic microsomal cytochrome P-450. Thismay cause a decreased metabolism of drugs known to utilize thisdegradation pathway. If a patient is on any medication known to bemetabolized by this pathway, appropriate monitoring procedures areundertaken.

Patients were given a 100-μg/0.5 ml vial of INF-γ given via I-Neb®(Philips Respironics) three times per week. Delivery to the lungs wasassessed via radiolabeled gamma camera studies. One hour aftermedication delivery, a peak flow reading was obtained and recorded.After the first aerosol treatment, each patient was required to remainon the unit for an additional four hours, when an additional lung examand peak flow measurement is taken. Each patient was monitored duringthe administration of INF-γ for side effects, including but not limitedto fever, fatigue, GI abnormalities, headache, cough, shortness ofbreath, wheezing, and laboratory abnormalities.

Toxicity was graded with “The Common Toxicity Criteria”. Dosemodifications are made accordingly. For Grade I toxicity, the patientmay continue treatment at the discretion of the physician. For Grade IItoxicity (confirmed by immediately repeating abnormal laboratoryparameters where appropriate) patient dose was held until a return toless than or equal to a Grade I toxicity, at which time the patient mayresume treatment. If Grade II or worse toxicity returns, the patient waswithdrawn from the study. For any Grade III or IV toxicity, the patientwas withdrawn from the study.

Example 3 Clinical Efficacy

A 38 year old Haitian woman with history of chronic allergies presentedwith a one and half year history of progressively increasing shortnessof breath and dyspnea on exertion. She was subsequently diagnosed assuffering from mixed connective tissue disease (MCTD). MCTD combinesfeatures of polymyositis, systemic lupus erythematosus, rheumatoidarthritis, scleroderma, and dermatomyositis, and is thus considered anoverlap syndrome. MCTD commonly causes joint pain/swelling, malaise,Raynaud phenomenon, muscle inflammation, and sclerodactyly (thickeningof the skin of the pads of the fingers). Distinguishing laboratorycharacteristics are a positive, speckled anti-nuclear antibody and ananti-U1-RNP antibody. It has been associated with HLA-DR4. The patient'sPFTs showed a predominantly restrictive pattern with low diffusioncapacity, suspicious for interstitial lung disease. She underwent a CTscan of her chest, which corroborated her PFT results, revealingsub-pleural fibrosis and honeycomb changes, predominantly at the lungbases. An open lung biopsy showed a pattern consistent with UIP/IPF.

Materials and Methods

The patient signed an informed consent approved by New York UniversitySchool of Medicine Human Subjects Review Committee. Aerosol INF-γ (400μg three times a week) was started in September of 2002 while thesubject continued to receive 10 mg of prednisone daily. The nebulizerAeroEclipse® (Trudell International, Canada) was filled with 200 μg ofINF-γ and normal saline solution to a final volume of 3 mL. The patientinhaled from the nebulizer using a relaxed pattern of tidal breathinguntil the device ran dry. It was refilled and the treatment repeated.The drug was administered at a flow rate of 8 L/min.

Pulmonary Function and Cardiopulmonary Exercise Testing

PFT and exercise testing were performed prior to starting INF-γ therapyand at the end of three months at New York University-Bellevue hospital.Subsequent PFT were performed at the discretion of the patient's privatepulmonologist at the Hospital for Joint Diseases.

Shortness of Breath Assessment

The University of California, San Diego (UCSD) shortness of breathquestionnaire (SOBQ) was completed by the subject prior to the start ofand at the end of three months of therapy with INF-γ. Used extensivelyin pulmonary rehabilitation, the SOBQ is a self reported 24-item measurethat assesses shortness of breath with various activities of dailyliving (ADLs). The patient indicated the severity of her shortness ofbreath on a six-point scale (0=not at all, to 5=maximal or unable to dodue to breathlessness) as related to 21 different ADLs with varyingdegrees of exertion. If a particular activity was not preformedroutinely, the patient was asked to estimate the anticipated degree ofshortness of breath. The SOBQ was scored by summing responses across all24 items to form a total score, which ranged from 0 to 122. Shortness ofbreath questionnaires have been shown to be a valid measure of healthimpairment in patients with chronic airflow limitation as well asrestrictive lung diseases, and to respond to change with therapy.

BAL

The patient underwent a research BAL in one of the radiographicallyinvolved segments of the lung prior to the start of therapy and at theend of three months. A flexible bronchoscope was used for the BAL, withadministration of xylocalne for local anesthesia. After six 50-mLaliquots of sterile saline solution were instilled, gentle suction wasapplied to allow recovery of bronchoalveolar fluid from aradiographically involved segment. The lavaged fluid was then filteredthrough two layers of gauze to remove mucus and centrifuged at 1,000revolutions per minute for 10 min. The fluid was concentrated (3 to the10×) [Centriprep-10; Amicon; Beverly, Mass.]. TGF-β was then measured bya purchased radioimmunoassay kit, in accordance with the manufacturer'srecommendations (R & D; Minneapolis). A microtiter plate reader was usedto determine concentrations. The sample was assayed in duplicate and theresults are reported as picograms per milliliter of BAL fluid.

Deposition Study

The patient signed an informed consent as approved by State Universityof New York (SUNY) at Stony Brook, Committee on Research Involving HumanSubjects. Details of the deposition technique were reported previously.(Condos et al., Chest 2004; 125: 2146-2155) Briefly, the patient wasplaced in a seated position in front of a gamma camera (Picker-Dyna 4C;Picker Corporation; Highland Heights. OH). For measurement of lungvolume and ventilation, xenon 133 (¹³³Xe) gas was employed. ¹³³Xe wasintroduced into a closed circuit of a “pulmonex”-Xenon trap (AtomicProducts; Center Moriches, N.Y.) during tidal breathing after which aposterior equilibrium image was obtained. The patient then inhaledradiolabeled INF-γ aerosol. Two serial depositions were performed, eachwith a 200 μg dose of INF-γ. A background image was taken prior to thesecond nebulization. The patient swallowed a glass of water immediatelyafter each nebulization, effectively washing any oropharyngeal activityinto the stomach. In order to measure the attenuation correction (AC)for the chest wall, a calibrated injection of a known quantity oftechnetium 99m (^(99m)Tc)-macroaggregated albumin (5 to 10 mCi) wasgiven through a peripheral intravenous line. Counts from a perfusionimage taken after the injection were subtracted from a background imageobtained prior to the injection. Net counts from the perfusion imagewere divided by the activity injected to yield an AC factor for thethorax (units=counts per min per microcurie). Analysis and storage ofall images was obtained using image processing software (Nuclear Power3.0.7; Scientific Imaging; Littleton, Colo.). The INF-γ aerosol particledistribution was measured using a protocol developed in the Stony Brooklaboratory. Mass median aerodynamic diameter was 2.2 μm.

Regional Deposition

Regions of interest were drawn over the ¹³³Xe equilibrium scan usingcomputer software. Three separate regions were identified. The wholelung zone was labeled as the region encompassing both lungs, and thecentral zone was labeled as the region over the large central airwayscomprising 33% of the area of both lungs. The peripheral zone is thearea which remains after deducting the central from the whole lung zone.These regions of interest overlying the ¹³³Xe equilibrium image werethen superimposed onto the ^(99m)Tc deposition images. The ratio betweencentral and peripheral (C/P) lung counts was calculated. In order tonormalize this ratio for differences in relative lung thickness the C/Pratio for ^(99m)Tc counts was divided by the C/P ratio for ¹³³Xe counts.This ratio defines the specific C/P ratio. A specific ratio of 1.0reflects deposition that is proportional to regional volume. A specificC/P ratio of unity reflects alveolar deposition as the central regionoutlines both central airways and the surrounding lung parenchyma.Increasing ratio in the C/P ratios greater than unity is consistent withincreasing deposition in the proximal airways.

Results

Objective findings are listed in Tables 1 and 2. PFT performed atinitial presentation revealed a predominantly restrictive pattern withreduced FVC (59% of predicted), TLC (53% of predicted), and DLCO/VA (51%of predicted). The patient was started on aerosolized interferon gammain September of 2002 and had multiple PFT done showing intervalimprovement in her DLCO/VA (from 51% to 68%) and stabilization of herFEV1 and TLC. Her exercise performance revealed increased maximal oxygenconsumption, decreased minute ventilation, and a reduction in the degreeof oxygen desaturation. The patient had significant improvement in herSOBQ measurement (decrease by 19 points). BAL fluid assayed for TGF-βone hour after treatment with aerosol INF-γ was substantially reduced incomparison to pre drug treatment levels (FIG. 1). The first depositionimage is shown in FIG. 2 (posterior scan). The AC value of the chest,measured by perfusion scan, was 184.4 counts per minute/μCi. Of the 400μg INF-γ placed in the nebulizer (two 200 μg treatments), 54.4 μg or13.6% was deposited in the lungs. Regions of interest are shown, derivedfrom ¹³³Xe equilibrium image and superimposed on the deposition image.The calculation of the specific C/P ratio, for deposition one and two,revealed ratios of 1.28 and 1.26 respectively. These ratios indicate arelative peripheral deposition (1.0 being the most peripheral possible).

The patient's chest CT showed no further progression in the bibasilarhoneycomb changes or sub-pleural fibrosis for more than one year.

Discussion

Recent studies implicate repeated alveolar and epithelial injury andassociated cytokine activation with resultant lung fibrosis. This caseillustrates the potential use of aerosol INF-γ as a novel mode oftherapy, bringing pharmacologic doses of the drug, far greater than thatwhich could be delivered by subcutaneous injection, directly to the siteof disease. At present levels of deposition in the lung parenchyma ourpatient demonstrated stabilization of her pulmonary function parameters.FVC and TLC<78% of predicted have been shown to have a negative impacton survival in patients with idiopathic interstitial pneumonias (Erbs etal., Chest 1997; 111: 51-57).

This patient had no significant decline in her FVC, and TLC over almosttwo years. She showed an improvement trend in her exercise physiologyreaching 46% of her maximum oxygen consumption. A substantial decreaseddyspnea score was noted at the end of 3 months as well as a significantdecrease in the pro-fibrotic cytokine, TFG-β.

TABLE 1 PFT results before and after therapy % PREDICTED DATE HISTORYTLC FEV1 FVC DLCO/VA April 2002 First PFT 53 49 59 51 July 2002 Afteropen lung Bx*; start of Tx† with 51 57 55 64 prednisone and azathioprineAugust 2002 Azathioprine D/C due to ↑LFT; prednisone 60 54 52 54 taperedto 10 mg/day; referred for research study; PRE Tx† PFT December 2002Post INF-γ Tx† 61 66 60 65 June 2003 Continued aerosol therapy 54 67 6161 BACK TO WORK December 2003 Continued aerosol therapy 57 61 53 71 July2004 Continued aerosol therapy 53 58 51 68 *Bx = biopsy; †Tx = treatment

TABLE 2 Results from exercise testing, dyspnea scores and musclestrength VARIABLE Pre Tx† Post Tx† (3 mo) V_(E,) max L/min 40.78 35.48VO₂ max, L/min  0.655 (42%)  0.746 (46%) Minimum O₂ saturation (%) 35 54UCSD SOBQ* 63 44 *University of California at San Diego, Shortness ofBreath Questionnaire †Tx = treatment

Example 4

BAL fluid is used for protein determination and assay of INF-γ using aviral inhibition assay to determine the amount of drug delivered.Concentrated BAL fluid and 24 hour cell culture supernatants are assayedfor cytokines IL-113, IL-4, IL-6, IL-8 and TNF-α by ELISA (R&D,Minneapolis). Cell-free BAL supernatant is used to measure TGF-βactivity by ELISA and luciferase reporter assay. Transbronchial biopsy(TBBX) specimens are used to measure TGF-β gene transcription bysemi-quantitative RT-PCR. Fibroblasts are obtained from TBBX specimens,and the quantities of collagen I, III, and fibronectin RNA measured byRT-PCR. RNA (10 μg) is obtained from TBBX or cell culture of TBBX, andNorthern Blot analysis is performed. Hydroxyproline protein content ismeasured by spectrophotometry using BAL fluid, BAL supernatants, andTBBX specimens. BAL fluid cell counts are calculated for each patient,in both pre- and post-treatment samples. A blood sample from eachpatient is obtained for storage.

Example 5

Five usual interstitial pneumonia (UIP) or IPF patients were studied.Each patient was asked to participate in a deposition study (underseparate consent) of INF-γ administered via hand-held nebulizer. Thisdeposition study was designed to study aerosolized INF-γ as follows. Thedrug was labeled with 99 mTc and administered via aerosol nebulizer.Using the “attenuation technique”, the dose of INF-γ delivered tovarious regions of the lung was calculated. The initial dose of 500 μgINF-γ was used, as this dose has previously been shown to be safe. Thedose is adjusted according to deposition studies in each individualpatient. A follow up bronchoscopy was performed at the end of thetherapy, using the protocol described above. BAL was guided by lungdeposition images, so that the areas of highest drug deposition wasanalyzed and compared to areas of lowest delivered drug and pre-aerosolINF-γ samples. In this way, total dose to each area of the lung can becalculated and determined. Depending on clinical response and BAL data,dose may be adjusted to reflect optimal clinical and depositionparameters. Attempts will be made to sample similar segments pre- andpost-treatment, when possible. Each patient has a follow up evaluationat one month post therapy. The results of all procedures, laboratoryevaluations, radiological studies, and pulmonary physiology evaluationsare documented in the patient's medical record. All study evaluationsare conducted at the GCRC of NYU Medical Center.

One commercially available breath-actuated nebulizer was used in thisstudy, the AeroEclipse, whose particle generation is dependent onpatient breathing through the nebulizer. It produces aerosol only duringinspiration.

INF-γ was radiolabeled using ^(99m)Technetium diethylenetriaminepenta-acetic acid (^(99m)Tc-DTPA) for both in vitro and in vivostudies. For AeroEclipse, 2 vials (250 mg of INF-γ) were used to make upa final volume of 2 mL. AeroEclipse was operated using a Pari Master aircompressor (PARI Respiratory Equipment, Inc. Monterey, Calif.)

The nebulizers were connected to the circuit in the manner of theirclinical use. A ten stage, low flow (1.0 Urn) cascade impactor(California measurements, Sierra Madre, Calif.) was connected using a Tconnector (T connector_(cascade), Hudson Respiratory Care, Temecula,Calif.). An inspiratory filter, that prevented particles from enteringthe cascade impactor during expiration, was placed between the pistonpump and cascade impactor. A second filter (leak filter) was placed inthe system to capture the excess particles directed neither to theinspiratory filter nor to the impactor. To assess possible effects ofpatient ventilation a piston pump (Harvard Apparatus, Millis, Mass.) wasused to simulate a patient's breathing effort.

Prior to inhalation the aerosol was studied on the bench under twoconditions:

Standing cloud: The cascade impactor sampled the particles directly fromthe tubing at 1 Lpm without any ventilation generated by the piston pump(pump disconnected from circuit). For the purpose of generation ofparticles from AeroEclipse, the breath actuation valve was pressedmanually for the duration of sampling.During Ventilation The Harvard pump was used to generate a sinusoidalflow in the system, analogous to the breathing of a patient. A tidalvolume of 750 mL; Respiratory Rate of 20/m and Duty Cycle of 0.5 wasused.

Aerodynamic particle distributions were measured as well as depositionon the connecting tubing to the cascade (T connector_(cascade)). Theballistic properties of the aerosol were quantified as the activity onthe T connector_(cascade) and reported as a percentage of the activitycaptured in the cascade impactor (% Cascade). This deposition was usedin predicting lung deposition.

Xenon imaging and attenuation studies For all the subjects INF-γdeposition was studied using the AeroEclipse nebulizer. Xenon imagingand attenuation studies (see below) were performed.

Lung volume and outline studies (¹³³Xenon (¹³³Xe) equilibrium scan) Thepatient was seated in front of a posteriorly positioned gamma camera(Picker Dina camera; Northford, Conn.). After taking a room backgroundimage for ^(99m)Technetium (^(99m)Tc), the camera was set for ¹³³Xe.Breathing tidally at functional residual capacity (FRC), the patientinhaled 5-10 mCi of ¹³³Xe until the count rate became stable ±10% over15 seconds. A 1.0 min gamma camera image (¹³³Xe equilibrium image) wasacquired and stored in a computer (Nuclear Mac v1.2/94; ScientificImaging Inc. Littleton, Colo.) for analysis. This image was used todefine the outer margins of the lung.

Aerosol deposition studies After ¹³³Xe imaging, the camera was switchedto ^(99m)Tc. Then, the patient inhaled radiolabeled aerosolized INF-γfrom the nebulizer. For each device an expiratory filter was present tocapture exhaled particles. The nebulizers were run until dry. Afterfinal inhalation, the patient drank a glass of water to wash materialfrom the oropharynx to the stomach. Measuring stomach activity assessedupper airway deposition.

Lung attenuation studies (perfusion scan) Lung perfusion scanning wasdone to calculate the attenuation factor of the lungs. Immediatelyfollowing deposition imaging, 5 mCi of ^(99m)Tc-albumin macroaggregateswere injected via a peripheral vein. It was assumed that all themacroaggregates traversed the right side of the heart and distributed inthe lung proportionately to regional perfusion. A one-minute image wasobtained. Perfusion was calculated as measured activity minus theactivity measured on the previous (deposition) image. The lungattenuation factor was measured by dividing the amount of activitymeasured by the camera by the amount of activity injected. Lungattenuation factor=Activity measured/activity injected

Stomach Attenuation The patient was given bread with a known amount of^(99m)Tc applied to it and a gamma camera picture of the stomach wastaken after ingestion. Stomach attenuation was calculated by dividingthe activity ingested by activity measured by the gamma camera. Stomachattenuation factor=Activity measured/activity ingested

Quantification of deposition Using the computer, regions of interestwere visually drawn around the stored equilibrium ¹³³Xe equilibrium scanto define the lung outline and encompass the lung volume. Central lungregions were then drawn that outlined the inner one third of thetwo-dimensional lung area. After the xenon regions were defined, thesame regions were placed over the deposition image and stomach activityidentified. Then, a “stomach region” was visually drawn outlining thestomach. If there was overlap between the stomach region and the xenonequilibrium region of the left lung, the overlapping region was definedas “stomach on lung” or SOL. For determination of whole lung deposition,radioactivity from the stomach and the stomach on lung regions wereexcluded.

Lung deposition was measured using the gamma camera by quantifyingactivity in the lung regions and applying the appropriate attenuationcorrection. Oropharyngeal deposition was determined by subtracting thelung activity from the total activity on the deposition image.Appropriate corrections were made for stomach attenuation.

Specific Central to Peripheral ratio (sC/P) Specific central toperipheral lung activity was defined by dividing the aerosol image bythe xenon equilibrium image. This ratio represents the distribution ofdeposited aerosol normalized for regional lung volume. sC/P for aerosoldeposition=(C/P aerosol/C/P xenon). If the aerosol behaves perfectly asa gas and follows the ¹³³Xe distribution, the sC/P ratio should be 1.0.Particles that deposit preferentially in central airways yield sC/Pratios of 2.0 or higher.

Results of Deposition study show significant deposition of aerosolthroughout the lungs. When normalized for lung volume, there arerelatively more particles in central lung regions than peripheral (sC/Pratio=1.618. There is minimal upper airway deposition.

Example 6 Effects of Aerosol INF-γ

Adverse effects We treated 15 individuals (normal volunteers andpatients with pulmonary tuberculosis) with aerosolized INF-γ. Theaerosol administration was well tolerated with few patients complainingof occasional cough or myalgias. The longest period of administrationwas 3 months without an increase in adverse effects. In addition, Jaffefound that aerosolized INF-γ given to normal subjects was safe, withoutsystemic side effects, and was able to activate alveolar macrophages andnot PBMC, as opposed to parenterally delivered r INF-γ, the effects ofwhich could only be noted in the peripheral blood (Jaffe et al., (1991)J Clin Invest 88(1): 297-302).

Deposition studies We investigated the aerosol depositioncharacteristics of INF-γ. A deposition image reveals that radioactivity(aerosol) is deposited to all normal areas of the lung. Disease andcavitary areas are spared. Perfusion scan shows minimal perfusion tocavitary areas as well. Preliminary determination of deposition revealsa range of 10-20% of aerosol dose delivered to the lung, using bothmass-balance technique and xenon (figure). We concluded that targeteddelivery of drug to the lung results in drug deposition in normal lungparenchyma (Condos et al., (1998) Am J Respir Crit Care Med 157(3):A187).

Bronchoalveolar lavage findings We previously demonstrated clinicalimprovement in a group of patients with severe multi-drug resistanttuberculosis treated with INF-γ. The patients underwent bronchoscopywith BAL of the radiographically involved area before and aftertreatment. 24-hour cell culture supernatants and fluid from the BAL wereassayed by ELISA and were found to have decreasing levels over time ofTNF-a (mean 172 to 117 pg/ml), IL1-b (mean 25 to 8 pg/ml) and noappreciable levels of INF-γ (mean 3.3 to 2.5 pg/ml). We concluded thatINF-γ administration is associated with a decrease in TNF-a producedlocally at sites of disease. This may in part explain the beneficialeffects of INF-γ in advanced in advanced MDR-TB (Condos et al., (1998)Am J Respir Crit Care Med 157(3): A187).

Example 7 Successful Treatment of Idiopathic Pulmonary Fibrosis

In a study of aerosol rINF-γ for five patients with usual interstitialpneumonia (UIP) or IPF, we found the treatment to be well tolerated.Adverse effects included fatigue, cough, and low grade fever (n=1).Routine laboratory assessment during the study period did not reveal anyabnormalities. All patients reported subjective improvements in theirshortness of breath. By the end of three months of treatment, patientsin the study had a statistically significant increase in total lungcapacity. FIG. 7 demonstrates the increased percent predicted total lungcapacity after treatment in three of the five patients treated. Therewas also an improvement of greater than 200 cc's (200 and 500 cc,respectively) in the Forced Vital Capacity in two of the five studypatients. FIG. 8 demonstrates the increased percent predicted forcedvital capacity after treatment in three of the five patients treated.These physiologic changes were accompanied by decreases in the levels ofactivated TGF-β recovered from broncheoalveolar lavage (BAL) fluid(fluid washed from the inside lining of the lungs) of these patients.FIG. 9 demonstrates the reduced portion of TGF-β of total protein in thefive patients treated. TGF-β is one of the key mediators of fibrosis inthe lung. Its activation leads to collagen production. Decreases in itslevels should lead to less collagen deposition and less fibrosis in thelung. In addition, we measured levels of interferon-γ in the BAL fluidof the patients before and after aerosol therapy and found an increaseassociated with aerosol administration of the drug. FIG. 10 demonstratesthe amount of interferon-γ measured in the lungs of tuberculosispatients and patients with idiopathic pulmonary fibrosis both before andafter aerosol treatment with interferon-γ.

In contrast to subcutaneous studies previously performed, we were ableto show that there was a physiologic improvement in lung function withaerosol delivery of rINF-γ. This improvement occurred over a treatmentperiod of three months compared to the one year treatment received bythe patients in another subcutaneous trial. This physiologic improvementwas associated with increases in levels of INF-γ in the lung leading todecreases in levels of activated TGF-β recovered from the lungs ofpatients after aerosol treatment. This data demonstrates the ability todeliver a pharmacologically important amount of interferon-γ to thelung. No detectable lung levels of interferon-γ were measured followingsubcutaneous administration. (See, Jaffe et al., J Clin Invest. 88,297-302 (1991). In an effort to further define lung dose, two of thefive patients had deposition studies performed. These studies confirmeddeposition of approximately 40 mcg of rINF-γ to the lung periphery. Nomeasurements of lung dose or lung levels of rINF-γ were measured orreported the subcutaneous rINF-γ trial.

Example 8 Cytokine Gene Regulation

In this study investigation of transcription factor abundance,phosphorylation, and DNA binding activities test the hypothesis thataerosol INF-γ treatments impinge on cellular signal transductionpathways to activate latent STAT-1 and induce de novo synthesis ofIRF-1. We performed these experiments on BAL cells obtained fromuninvolved and involved areas of lung in patients with pulmonary TB preand post treatment with INF-γ (Condos et al., (1999) Am J Respir CritCare Med (in press)). Purifying and cloning IRF-1 was a principal partof the initial work performed by Richard Pine, Ph.D., in the Laboratoryof Molecular Cell Biology at Rockefeller University with James E.Darnell, Jr. (Pine et al., (1990) Mol Cell Biol 10(6): 2448-57).Immunoblot and electrophoretic mobility shift assays the same as orsimilar to those proposed for Aim 3 of this project have been employedin the work mentioned here.

The results of cytokine gene manipulation in the uninvolved lungs oftuberculosis patients are most relevant Results show that in both theadherent (mainly alveolar macrophages) and the nonadherent (lymphocytesand polymorphonuclear cells) portions of the BAL cells, there is anincrease in the amount of specific IRF-DNA and STAT-1-DNA complexesafter aerosol INF-γ treatment.

Example 9 Efficacy in Asthma Treatment

We will recruit 30 patients with mild to moderate asthma to receiveINF-γ aerosol versus standard treatment. The study will be performed asa randomized, placebo controlled, cross-over, double-blind r INF-γaerosol delivery study in subjects with mild-moderate persistent asthmarequiring moderate dose inhaled corticosteroid for symptom control.

Patients must be between the ages of 18 and 65 yr., any race or sex.They must be current nonsmokers with <10 pack year history of cigarettesmoking. Patients who meet NAEPP guidelines for a diagnosis of asthmawill be enrolled. We will recruit patients with mild-moderate persistentasthma, with baseline forced expiratory volume in one second (FEV1)greater that 70% of predicted value and evidence of reversibility (≧15%improvement in FEV1 post-bronchodilator treatment). These patients willbe required to be on intermittent use of inhaled B2-agonists and lowdose inhaled corticosteroids. Low dose inhaled corticosteriod useincludes 168-500 mcg/day of beclomethasone dipropionate, 200-400 mcg/dayof budesonide DPI, 500-1000 mcg of flunisolide, or 400-1000 mcg/day oftriamcinolone acetonide.

Patients who are pregnant, have contraindications to fiber opticbronchoscopy, are current smokers, or have a >10 pack year history ofcigarette smoking will be excluded. Any patient with a history of poorlycontrolled or severe asthma, history of recent systemic corticosteroiduse, or history of recent exacerbation or infection will also beexcluded.

We will incur a 1 month “wash-in” period to allow all recruited patientsto start at the same baseline dosages of inhaled corticodsteroids(beclomethasone dipropionate 4-12 puffs/day). Each patient will have atbaseline:

1) Complete history and physical examination and routine laboratorywork,2) Pulmonary function measurements (FEV1, FVC, and PEFR),3) 50 ml heparinized blood drawn by venous stick,4) Blood samples will be obtained for total IgE, specific IgE to definedallergens, and eosinophil count,5) Fiberoptic bronchoscopy with BAL, with analysis of cellcount/differential and levels of INF-γ, IL-4, IL-5, GM-CSF, IL-10,IL-12, and IL-13 by ELISA of 24 hour culture supernatants.

We will then administer aerosol r INF-γ (500 mcg) 3 times a week for 8weeks to 15 patients. Equivalent amounts of aerosolized saline will beadministered to 15 patients in a randomized fashion. At eight weeks wewill allow for a 1 month wash out period and cross over the subjects tothe second arm of the trial.

The patients will have at each treatment visit:1) Brief questionnaire regarding signs and symptoms.2) Review of daily diary cards and monitor use of b-agonists.3) Peak flow monitoring before and after aerosol treatment4) Abbreviated history and physical exam performed weekly.5) Subjects will characterize symptoms in a daily diary before, during,and after aerosolized INF-γ treatment. They will rate symptoms of cough,wheeze, and shortness of breath on a scale. They will also record dailypeak flow measurements.

At completion of each arm of the trial (at eight weeks of eitheraerosolized INF-γ treatment or control saline) subjects will have:

1) Complete history and physical examination and routine laboratorywork.2) Pulmonary function measurements (FEV1, FVC, and PEFR)3) 50 ml heparinized blood drawn by venous stick.4) Blood samples will be obtained for total IgE, specific IgE to definedallergens (RAST), and eosinophil count.5) Fiberoptic bronchoscopy with BAL will be performed the day after thelast INF-γ treatment or no treatment. We will analyze BAL cellcount/differential and levels of INF-γ, IL-4, IL-5, GM-CSF, IL-10,IL-12, and IL-13 by ELISA of 24-hour culture supernatants.6) All patients will continue to be followed monthly in the asthmaclinic:

a) complete history and physical

b) routine labs

c) spirometry and peak flow measurements

We have chosen to study patients with mild-moderate persistent asthma onlow dose inhaled steroids for a variety of reasons. First, to includeonly patients with mild intermittent symptoms may not allow us thesensitivity of finding physiologic, symptomatic, or immunologic changesin such a population. Second, we cannot allow any of our patientsrequiring inhaled steroids to discontinue treatment for this trial. Inaddition, the purpose of this trial is to determine if aerosolized INF-γcan serve as an adjunct to already specified treatment regimens. Weunderstand that the use of inhaled steroids may confound our study ascorticosteroids can affect cytokine levels. We will include a “wash in”period to start all subjects at the same baseline.

Example 10 Effect on Pulmonary Function of Asthmatics

To determine the effects of aerosol r INF-γ on pulmonary functionmeasurements, we will obtain spirometry values of forced expiratoryvolume at one second (FEV1), forced vital capacity (FVC), peakexpiratory flow rate, and lung volumes including total lung capacity(TLC), and functional residual capacity (FRC) for each subject prior toaerosolized treatment. These will be performed in the Bellevue HospitalPulmonary Function Laboratory.

We expect to find a mild improvement in peak flow measurementsimmediately following administration of aerosolized r INF-γ, as we havenoted in treating tuberculosis patients who did not have asthma.Currently, it is unclear why INF-γ would have a bronchodilator effect.We also expect an improvement in FEV1, and FVC after 8 weeks of aerosolINF-γ treatment, reflecting reduced airway inflammation.

We choose to follow FEV1 particularly because it is reproducible forindividual patients. These values are specific for large airwayobstruction. Should we find during the initial portion of the study thatother variables of small airways disease is affected, we will usespecific airways conductance, airways resistance, or forced expiratoryvalues at 25-50% as endpoints. Should more sensitive tests of airwayresistance be required, we may perform frequency dependence ofcompliance studies. We can also perform bronchial provocation studieswith methacholine to study airway hyperreactivity. These additionalstudies are not as reliable because of individual variability as well asinterindividual variability. The study design as a cross-over trialshould avoid interindividual variability.

Example 11 Effects on BAL Specimens

BAL specimens will be obtained from the 30 asthma patients. We willadminister aerosol INF-γ to 15 of these patients for 8 weeks in order toassess whether INF-γ modulates cytokine production. These patients willhave pre- and post-treatment BAL and blood draws.

Methods Fiberoptic bronchoscopy Subjects will be pre-screened withmedical history and physical examination, spirometry, oximetry,assessment of bronchial hyperresponsiveness, coagulation tests (PT, PTT,platelets), and CBC and screening chemistries. During the procedurepatients with have continuous monitoring of heart rate and O₂saturation, recording of subject symptoms and medication doses,intravenous catheter in place, premedication with inhaled b-agonist,subcutaneous atropine (0.4 mg), and sedation (midazolam, iv), andsupplemental oxygen. The fiberoptic bronchoscope is introduced afterlight premedication and topical anesthesia of the nose and upper airway.The tip of the bronchoscope is wedged into a segmental, or subsegmental,bronchus of the right middle lobe or lingula. One hundred milliliters of37° C. normal saline are instilled into the bronchus in aliquots of 20ml. The warmed saline should avoid thermally induced bronchospasm inasthma subjects. Gentle intermittent suction is used to recover theeffluent. Fluid recovery of 60 to 80% is expected in mild asthmatics.Recovery is reduced to 50% in subjects with moderate to severe disease[Jarjour, 1998 #62]. Pulse oximetry and clinical assessment of patientstatus will continue post-procedure. Discharge instructions will includefollow-up appointment within the week, and contact telephone number.

Alveolar macrophages (AM) and BAL cells Cells will be collected bybronchoalveolar lavage (BAL) performed by standard techniques, andprepared for culture as follows: The fluid is filtered through one layerof sterile gauze to remove clumps of mucus. A total cell count is donein a hemocytometer and cell differentials performed on cytocentrifugeslides stained with modified Wright-Giemsa stain with a total of 500cells counted. Cell viability is determined by Trypan Blue exclusion,and in all cases recovered cells to be used for experiments will begreater than 90% viable. Twenty cytocentrifuge slides will be preparedfrom each lobe of BAL and once fixed in 10% formalin, frozen at −70° C.BAL cells will be washed and cultured (37° C.) in RPMI (GIBCO)supplemented with 10% heat-inactivated fetal calf serum (FCS) and 100u/mL penicillin and 100 mcg/ml streptomycin at a concentration of 10⁶cells/ml for 24 hours.

Peripheral blood Blood will be obtained by venous stick at a constanttime in the day, before and after completion of INF-γ treatment. PBMCswill be isolated from heparinized venous blood by Ficoll-Hypaque densitygradient centrifugation. Heparinized venous blood is layered onFicoll-Hypaque and centrifuged at 2500 rpm for 20 minutes. The lowdensity layer of PBMCs will be aspirated and washed with phosphatebuffered saline (PBS) and resuspended at a concentration of 10⁶ cells/mlof RPMI-1640 (GIBCO) with 10% heat-inactivated FCS, 100 U/ml penicillin,and 100 mcg/ml streptomycin. The cell cultures will then be incubated at37° C. and 5% CO₂ for 24 hours. The cell supernatants will then becollected and assayed for cytokines by ELISA.

Serum samples will also be collected to determine specific IgE (RAST) toallergens associated with urban asthma (D. pteronyssinus, D. farinea, B.germanica—German cockroach, and P. americana—American cockroach).

An additional study will be performed on PBMCs obtained from atopicasthmatics and normal controls. This will entail PBM cell culture afterisolation as described above in the same supplemented RPMI culturemedia. These cultured cells will then be stimulated with a nonspecificstimulus (LPS) or with a known allergen. The culture supernatants willthen be assayed for cytokines by ELISA. These levels will be compared toresting cell cytokine levels. This evaluation can be done to screen alarge urban population of asthma subjects for recruitment of patientswith a baseline poor INF-γ response into the aerosolized INF-γ treatmenttrial.

Assessment of cytokines We will assay BAL cell supernatants collectedover 24 hours at 10⁶ cells/ml by ELISA (Endogen) for INF-γ, IL-4, IL-5,IL-10, IL-12, IL-13 and GM-CSF. We collect 5 tubes of 10⁶ cells/ml sothat we can run samples in triplicate for each cytokine. Since weaverage 30 to 40×10⁶ BAL cells per lung segment, we can expect toevaluate BAL cell supernatants for each patient. We will not measurecytokines in BAL fluid, since we may retrieve INF-γ in the post-BALspecimens, and our interest is the release of cytokines from BAL cellsspontaneously.

Example 12 Mechanisms of Gene Regulation Affected by INF-γ Treatment

The clinical treatment protocol will have clear-cut effects on theabundance and activity of transcription factors that regulate geneexpression in response to INF-γ and that correlation of these data withthe cytokine profile will extend the criteria by which the immuneresponse in asthma can be evaluated. Furthermore, the data obtained willallow mechanistic interpretation of the results from analysis ofcytokine production and expression of cytokine and other genes.

The design of the project incorporates several controls to helpestablish the effect of the aerosol INF-γ treatment, distinct from anyother variable. These include obtaining BAL and blood samples before andafter the course of treatment, and collection of BAL samples fromuninvolved as well as involved lobes. All experiments for this aim willbe done with protein extracts prepared from BAL or PBM cells.Cytoplasmic and nuclear proteins will be obtained and analyzedseparately. To gain more definitive results, BAL cells will be separatedinto adherent and nonadherent populations. The former will includepredominantly alveolar macrophages. The latter will be comprisedpredominantly of lymphocytes and granulocytes. PBMC will be extractedwithout further separation.

Investigation of transcription factor abundance and DNA-bindingactivities for this project will test the hypothesis that the clinicalprotocol of aerosol INF-γ treatments will impinge on cellular signaltransduction pathways to activate latent STAT-1 and induce de novosynthesis of IRF-1 and CIITA. The data obtained will relate themolecular mechanisms that regulate gene expression to initial clinicalobservations. The results from a limited course of therapeutic in vivotreatment with aerosol INF-γ will have far greater predictive power fordesign of future trials when interpreted in conjunction with the datathat demonstrate the molecular response to the therapy.

Determine abundance of transcription factors STAT-1, IRF-1 and CIITAThere are two major reasons why it will be valuable to quantify thetotal amount of these transcription factors before and after thetreatment protocol. These data will be critical for overallinterpretation of the regulated response to the INF-γ treatments. Itwill lead to conclusions about the extent to which the available proteinis subject to phosphorylation events and the proportion of total proteinthat has DNA-binding activity. Additionally, the abundance of theproteins is the final measure of regulated expression of the genes thatencode the factors and thus provides a foundation for future studies tofurther integrate the functional and regulatory aspects of the evokedimmune response. Immunoblot detection will be the primary techniqueused. Cytoplasmic or nuclear extract from up to 5×10⁶ cells will be usedfor each analysis. Obtaining cells as described above will yield 10samples for each patient. All the extracts of PBMC and BAL cells fromone patient will be included in a single experiment, which willfacilitate relative quantitation within a set of samples. Controlcytoplasmic and nuclear extracts prepared from cultured cell lines willalso be included in each experiment. On the basis of previous studies,these samples will be known to contain the target proteins, and can thusprovide positive controls for the immunoblot detection. Additionally,they can be used to validate that the data obtained are quantitative orreveal the limits of the quantitative detection.

The proteins will be separated by SDS-PAGE, then transferred to amembrane. The membrane will be developed with reagents to detect STAT-1,IRF-1, and CIITA, one after the other. The membrane will be probedfinally to detect b-tubulin, which will be present in both cytoplasmicand nuclear protein extracts and can thus serve as an internal standardfor quantitative comparison of cytoplasmic or nuclear extracts withinand between experiments. All the antibodies needed are available in thelaboratory or can be commercially obtained and are known to work forimmunoblot protocols. To allow the sequential detection of differentproteins, the membrane will be treated to disrupt antibody bindingwithout releasing the target proteins. This approach can be proven towork by repeating the detection of each target protein in sequence, andcomparing the signals obtained in the first and second round. Negativecontrols for specificity of detection are provided for each protein bythe antibodies against the other two. Additionally, the membrane will bedeveloped a final time without inclusion of a primary antibody.

If the number of cells available is insufficient, or the abundance of aparticular transcription factor is too low, a signal will not bedetected. It might be possible to gain greater sensitivity by employingELISA assays for these proteins, but the greater reliability andspecificity of the immunoblot method would be lost. However, neither ofthese potential problems is likely to be significant. The desired numberof cells should routinely constitute only a fraction of each BAL sample.Low abundance of any target protein would be a physiologically relevantresult leading to meaningful conclusions. However, it should be notedthat the reagents and detection systems available will provide a signalif there is 100-1000 copies of the target protein per cell, which wouldcorrespond to not more than 8 femtomoles (0.5-1.5 ng) in the analyzedaliquot.

Characterize tyrosine and serine phosphorylation of STAT-1, IRF-1 andCIITA Changes in transcription factor phosphorylation are often the linkfrom the presence of the factor to its function. This is true even ifDNA-binding activity is not directly altered by changes inphosphorylation (David et al., (1995) Science 269(5231):1721-3; Wen, Z.et al., (1995) Cell 82(2): 241-50; Pine et al., (1994) Embo J 13(1):158-67; Cho et al., (1996) J Immunol 157(11): 4781-9; David et al.,(1996) J Biol Chem 271(27):15862-5; Gupta et al., (1995) Science267(5196):389-93; Hibi et al., (1993) Genes Dev 7(11): 2135-48; Parkeret al., (1996) Mol Cell Biol 16(2): 694-703; Schindler et al., (1992)Science 257(5071): 809-13; Shuai et al., (1992) Science 258(5089):1808-12). As described above, both tyrosine and serine phosphorylationof STAT-1 are regulated and control its activity. IRF-1 is aphosphoprotein, but naturally occurring changes in phosphorylation havenot been documented, and phosphorylation of CIITA has been littlestudied. The experiments described here will provide data on the in vivoregulation of STAT-1 by examining the extracts from PBMC and BAL cellsfor phosphorylation events previously documented primarily incell-culture systems. The data obtained on IRF-1 and CIITA will gobeyond what has been determined previously.

The most straightforward design for this set of experiments is toquantitatively recover the target proteins from cell extracts byimmunoprecipitation, separate the recovered proteins by SDS-PAGE, thendetect phosphorylation by immunoblot analysis of the separated proteins.It is well established that commercially available anti-phosphotyrosineantibodies can be used to develop immunoblots and determine the presenceand extent of tyrosine phosphorylation, with little or no dependence onthe particular target protein. Antibodies against phosphoserine are alsocommercially available, but it is not certain that they will detect suchresidues in the intended target proteins. Thus, there is someuncertainty as to the successful application of this approach. Inaddition to the types of positive and negative controls described above,specific detection of phosphotyrosine or phosphoserine can be shown byincluding the phosphoamino acid in solution and observing that signalsare not obtained.

Although the proteins denatured by SDS-PAGE are most likely to reactwith the antiphosphoserine antibodies, it remains possible that ELISAwould be a successful alternative if immunoblot detection of serinephosphorylation does not work. In that case, wells would be coated withantibody to the target protein, the protein would be bound, and thedetection step would utilize antiphosphoserine primary antibodiesobtained from a different species than the source of the antibodiesagainst the target proteins. The controls utilized for the immunoblotwould essentially apply as well to an ELISA system.

Another alternative is available for analysis of STAT-1 serinephosphorylation. A specific anti-phosphoSTAT-1 (P-Ser) antibody could bemade (see Methods) based on the known position of the serine that issubject to regulated phosphorylation, Ser 727 (Wen, Z. et al., (1995)Cell 82(2): 241-50). This is quite likely to succeed, since commerciallyavailable antibodies specific for phosphorylated forms of severalproteins (New England BioLabs) have been obtained by immunization withthe appropriate phosphopeptide followed by purification of the specificantibody by use of protein-a, peptide, and phosphopeptide affinitymatrices. A similar approach would be used for this project. Metaboliclabeling of cells with ³²P-orthophosphate followed by specificimmunoprecipitation of target proteins and phosphoamino acid analysis isnot appropriate for this project, since the target proteins could bemodified during the culture period necessary for the metabolic labeling,and because the amount of material available is not likely to besufficient for such an approach. While determination of changes inserine phosphorylation may not be readily achieved for any of the targetproteins, the likely significance of that regulated post-translationalmodification strongly supports making the attempt.

These data will be used to establish the connection between theabundance of these factors and their function in this system. The extentof STAT-1 tyrosine phosphorylation will determine the level ofactivation and thus set minimum and maximum levels of DNA-bindingactivity. Changes in serine phosphorylation may modulate the DNA-bindingactivity and have additional effects on STAT-1 function as describedabove. It is possible that increased abundance of STAT-1 and basallevels of phosphorylation will be detected. Such a result would implythat the in vivo response overall is similar to the response of culturedcells exposed to INF-γ for a prolonged period and reflects the timecourse of post-translational modifications (phosphorylation anddephosphorylation) together with de novo synthesis of STAT-1 asmolecular responses to INF-γ. Data on changes in IRF-1 or CIITAphosphorylation will serve as additional markers of in vivo molecularresponses to INF-γ and will provide a strong rationale for future basicresearch to determine the functional significance of such changes.Should there be changes in abundance but not phosphorylation, it may bethat phosphorylation of these factors is not regulated in this system,or, as is possible for STAT-1, that there was no net change thatpersisted at the time the samples were obtained. Future studies in othersystems would be necessary to distinguish these possibilities.

Measure DNA-binding activity of STAT-1, STAT-4, STAT-5, STAT-6, andIRF-1 Measurement of DNA-binding activities will provide the final dataneeded to evaluate regulation of the molecular responses to thetreatment protocol. Detection and quantitation of the STAT family andIRF-1 transcription factors by electrophoretic mobility shift assay ofexperimental samples will be accomplished by established procedures.Extracts prepared from cultured cells will be included in these assaysas positive controls. Controls for specificity and identification of thefactors in question will be provided by performing reactions thatinclude competitor oligonucleotides or antisera. Both nonspecific andspecific oligonucleotides or antisera will be used.

In contrast to the synchrony of a cell-culture system, in which theentire population is exposed to an added cytokine starting at the sametime and lasting for the same amount of time, cells obtained by BAL orin blood samples will represent the total effect of repeated aerosolINF-γ treatment superimposed on the asynchronous start and duration ofexposure of individual cells governed by their trafficking into and outof sites where they are exposed. In cell-culture models, INF-γactivation of STAT-1 DNA-binding activity occurs within minutes. In somecell lines, the activity decays very rapidly. In others, including themonocytic cell lines NB4, U937, and THP-1, it persists for several hours(R. Pine and E. Jackson, unpublished). Since STAT-1 regulates the IRF-1gene, induction of IRF-1 DNA-binding activity by INF-γ is typicallydetected only after 1-2 hr, but then persists at least 16 hr. Thus,STAT-1 and IRF-1 DNA-binding activity may be present simultaneously, butit is also possible that only one or the other will be detected.

The results obtained from the experimental samples may reveal that bothSTAT-1 and IRF-1 DNA-binding activity are present, and thus wouldindicate that in vivo the net outcome of intermittent doses over severaldays is equivalent to an intermediate time of exposure in a cell-culturesystem. This would be distinct from the constitutive activation of STATfactors that has been reported in Bcr/abl-transformed cell lines or PBMCfrom leukemia patients (Carlesso et al., (1996) J Exp Med 183(3):811-20; Gouilleux-Gruart et al., (1996) Blood 87(5):1692-7).Furthermore, such a result would strongly suggest that the full panoplyof responses to INF-γ was ongoing at the time the samples were obtained.Alternatively, only STAT-1 or IRF-1 DNA-binding activity might bedetected. It seems unlikely that only STAT-1 will be detected, sinceprolonged induction of IRF-1 is the norm, while STAT-1 activation istypically transient. The presence of IRF-1 DNA-binding activity in theabsence of STAT-1 DNA-binding activity would imply that the treatmentevoked a response equivalent to those seen in cultured cells afterovernight treatment with INF-γ. Physiologically this would be consistentwith a situation in which the presence of INF-γ had persisted longenough to evoke biological endpoints such as monocyte to macrophagedifferentiation or elaboration of a Th1 T-cell response.

Assays of STAT-4, -5, and -6 will provide molecular markers for theresponse of T cells to IL-2, as well as the presence and function of keyTh1 or Th2 cytokines. Numerous recent reports have shown that IL-2activates STAT-5, IL-12 activates STAT-4, and IL-4 activates STAT-6 (Choet al., (1996) J Immunol 157(11): 4781-9; Gilmour et al., (1995) ProcNatl Acad Sci USA 92(23): 10772-6; Schindler et al., (1992) Science257(5071): 809-13). The data obtained here cannot themselves prove suchinterpretations, since almost every member of the STAT family isactivated by more than one cytokine, and almost every cytokine canactivate more than one STAT. Specifically, STAT-4 is also activated byINF-α, STAT-5 is also activated by IL-7, IL-15, prolactin, and growthhormone, and STAT-6 is also activated by IL-13 (Ivashkiv, L. B. (1995)Immunity 3(1): 1-4; Darnell (1996) Recent Prog Horm Res 51:391-403; Choet al., (1996) J Immunol 157(11): 4781-9). It should also be noted thatSTAT-1 can be activated by IL-6 and IL-10, as well as INF-γ. However,interpretation of this data would be supported by the analysis ofcytokine gene expression described above. Furthermore, the assays ofSTAT-4, -5, and -6 DNA-binding activity would significantly extend thoseobservations by providing data on intracellular molecular effects thatoccur in conjunction with a defined cytokine profile.

Methods We will extract mRNA from 10×10⁶ BAL cells using GITC andultracentrifugation. Since RT-PCR can be performed on such smallaliquots of cells, total RNA will be extracted, stored at −70° C., andassayed for gene expression of IRF-1. PCR primers will be based on thepublished sequences and utilize RT-PCR as described for cytokine genes,and compare transcript intensity to b-actin or GAPDH as a control. AsIRF-1 is basally expressed, a quantitative approach to RT-PCR will beneeded. Total RNA from BAL cells will be reverse-transcribed usingoligo-d(T) and PCR, according to standard methods. First-round PCR willbe carried out with 20% of the cDNA using the followingoligonucleotides: forward primer 5′_GTCAGGGACTTGGACAGGAG-3′, and reverseprimer 5′-AGCTCGGGGGAAATGTTAGT-3′. IRF-1 expression will be normalizedagainst GAPDH expression.

Preparation of cell extracts Cells from BAL will be processed into RPMImedia with no serum, as described above, then counted, then transferredto tissue-culture plates. After 2 hr at 37° C., nonadherent cells willbe removed with the media and counted again. The number of adherentcells will be obtained as the difference between the two cell counts.PBMC will be processed into RPMI media with no serum, as describedabove, then counted. All remaining steps will be carried out at 0-4° C.Cells in suspension will be centrifuged (200×g, 10 min), then thesupernatant will be aspirated and the pellet resuspended inphosphate-buffered saline (PBS). This step will be repeated, then thesecells will be centrifuged once more, and the final PBS supernatant willbe aspirated. Attached cell monolayers will be washed by adding thenaspirating PBS. PBS will be added again and the monolayers will bedetached by scraping. The cells and PBS will be transferred tocentrifuge tubes and centrifuged, and then the PBS will be removed byaspiration. Washed cell pellets will be lysed by suspending them inlysis buffer (20 mM Hepes.Na, pH 7.9, 0.1 mM EDTA.Na, 0.1 M NaCl, 0.5%NP-40, 10% glycerol, 1 mM DTT, 0.4 mM PMSF, 3 μg/ml aprotinin, 2 μg/mlleupeptin, 1 μg/ml pepstatin, 100 μM Na₃VO₄, 10 mM Na₂P₂O₇, 5 mM NaF) (3μl per 10⁵ cells) and incubating them for 5 min. Nuclei will berecovered by centrifugation (500×g, 10 min). The supernatant will beremoved, then clarified by centrifugation (13,000×g, 15 min). Theresulting supernatant will be recovered as the cytoplasmic extract,frozen in crushed dry ice or liquid nitrogen, then stored at −80° C. Thenuclear pellet will be resuspended in wash buffer (lysis buffer withoutNP-40), then recovered by centrifugation. The supernatant will beaspirated, then the pellet will be suspended in extraction buffer (washbuffer, except 0.3 M NaCl instead of 0.1 M) (3 μl per 10⁵ cells) andmixed for 30 min. The extracted nuclei will be pelleted bycentrifugation and the supernatant recovered as the nuclear extract,which will be frozen and stored as above. Protein concentrations will bemeasured so that comparable amount of different extracts can be used inan experiment. This usually entails using the same volume of eachextract, since the use of a fixed ratio of extraction buffer volume tocell number typically yields uniform protein concentrations for nuclearor cytoplasmic extracts within a single set of extracts and fromdifferent preparations.

Immunochemical procedures Immunoblots will be performed as follows:Extracts and protein size standards will be mixed with concentratedLaemmli sample loading buffer for SDS-PAGE, and applied to adiscontinuous Tris-glycine gel system prepared with an 8% separating geland a 4% stacking gel according to standard protocols. This gelpercentage will resolve all the proteins of interest. Electrophoresiswill be performed at constant voltage until the marker dye reaches thebottom of the gel. The gel will be equilibrated in transfer buffer(Tris-glycine plus 15% methanol), and then proteins will be transferredwith the same buffer to nitrocellulose membrane using a semidryapparatus (BioRad Transblot, S. Dak.). Membranes will be developed bystandard procedures. Briefly, this will entail blocking by incubationwith nonfat dry milk in Tris-buffered saline plus Tween 20 detergent,incubating with a specific primary antibody, washing several times inblocking solution, incubating with an enzyme-linked second antibody,washing in buffer without blocking agent, and incubating with an enzymesubstrate. For chemiluminescent substrates, signal will be detected withX-ray film. Alternatively, a Molecular Dynamics Storm 860 instrument isavailable at PHR1 for detection of signal from chemiflourescentsubstrates. Based on the experimental design, the optimal conditions fortransfer of STAT-1 and IRF-1, which were previously determined to beessentially the same (Pine et al., (1994) Embo J 13(1): 158-67; Pine etal., (1990) Mol Cell Biol 10(6): 2448-57; Pine unpublished), will beused for this project, as will the previously determined optimaldevelopment conditions for each of those proteins. For CITTA, optimaldetection conditions, including the choice of blocking agent, detergentconcentration, time of incubation for each step, and detection methodwill be empirically determined with control extracts prepared fromcultured cells. Immunoprecipitation of STAT-1 and IRF-1 will beperformed as previously described, with minor modifications.Specifically, the use of S. aureus cells for recovery of IRF-1 bound toanti-IRF-1 antibodies has been replaced by the use of protein-a agarose.

Should an ELISA assay be desired for detection of transcription factorabundance or to examine phosphorylation, detailed methods based onstandard procedures will be developed empirically with the use ofcontrol extracts from cultured cells. To increase sensitivity, thepreferred approach will entail binding of a capture antibody to thewells of a microtiter dish, followed by blocking, then incubation withthe desired extract. After further washing, the second specific antibodywould be used, and then the enzyme-linked antibody against the secondaryantibody would be used, and the substrate incubation performed. Washeswould be included after each antibody incubation. For STAT-1, it will bepossible to use rabbit polyclonal antiserum to provide captureantibodies and mouse monoclonal antibodies for detection, or vice versa,since both are available for the protein and for phosphotyrosine orphosphoserine. For IRF-1 and CIITA, only rabbit polyclonal antibodiesagainst the protein are available, so it will be necessary to use mousemonoclonal antibodies against phosphotyrosine or phosphoserine.Detection of those proteins will require that the extract be used tocoat the wells of the microtiter dish, followed by incubation withspecific primary antibody and enzyme-linked secondary antibody. Controlsfor specificity in assays of experimental samples will include omissionof primary antisera and/or inclusion of phosphoamino acids, asappropriate.

Electrophoretic mobility shift assays Optimal assays have been developedfor each of the indicated STAT family members and for IRF-1 (Pine andGilmour, supra). Reactions will include nonspecific and specificcompetitors, or nonspecific and specific antibodies. Each reaction willbe done with 5 μg of extract protein, which is typically 2-3 μl. Forreactions with competitors, those oligonucleotides will be included withthe radiolabeled probe when it is mixed with the extracts. For reactionswith antibodies, the protein-DNA-binding reactions will be carried outas usual, then antiserum will be added and the incubation continued.When incubations are completed, reactions will be applied to nativepolyacrylamide gels, which will then be electrophoresed at 4° C. Afterthe gels are dried, the results will be obtained by autoradiography, orwith a Molecular Dynamic PhosphoImager.

We will compare data obtained at baseline and after r INF-γ treatment byStudent's paired t test and express analysis as mean±SEM. Based onprevious studies, we will need to detect a 0.3 L difference in FEV1 anda difference of 3×10⁵ cells/ml. With 30 subjects, we will have a powerof 80%. Thus we will recruit 15 subjects for each group.

Subject Population Medical evaluations will be performed on 400individuals with asthma. Thirty patients with mild-moderate persistentallergic asthma will be randomized to receive INF-γ aerosol (n=15)versus standard treatment (n=15). Patients must have pulmonary functionand bronchial provocation measurements. There must be nocontraindication to fiber optic bronchoscopy. The majority of the studypopulation will be recruited from the Bellevue Hospital Primary CareAsthma Clinic. The demographic characteristics of our patient populationare: 90% minorities (mainly Hispanic and African American), aged 18-79(median=39), and have a 1:2 male:female ratio.

Potential Risks In general, the risk and severity of side effects tointerferon-γ (INF-γ) are related to the amount of medication given. Atthe dose used in this study (50 mcg/m²), most common possible sideeffects include fever, headache and malaise. Occasional nausea andvomiting have been reported at high doses. Aerosolization has not beenassociated with adverse reactions, although headache, cough and fevermay be expected. In the event of severe symptoms, medication will bestopped. There is a risk of previously unknown side effects of INF-γ notrelated to asthma. An individual may develop an allergic reaction to theprotein portion of INF-γ, in which case it will be discontinued.

Risk Management Procedures To minimize any risks, bronchoalveolar lavageis performed after medical evaluation excluding individuals with cardiacdisease or history of angina. Chest x-ray and blood studies includingbleeding parameters are performed. Bronchoalveolar lavage will beperformed by pulmonary fellows under faculty supervision. Following theprocedure, the study subjects will remain NPO for 3 hr and vital signswill be taken every 30 min for 3 hr. All patients will have cardiacmonitoring during the procedure and will receive nasal O₂ during andafter the procedure for 2 hr to prevent any hypoxemia. All patient datawill be kept locked in the pulmonary research offices. In the event ofadverse effects to subjects, a “crash cart” is kept with the fiber opticbronchoscope, including endotracheal tubes, injectable lidocaine andepinephrine, etc, and all procedures are done in the hospital with housestaff and a CPR team on call. All of the bronchoalveolar lavageprocedures will be periodically reviewed to identify any increasedincidence of untoward effects and identify their cause.

Example 13

All patients receiving aerosol interferon-γ were studied with spirometryto assess reversible airways disease. Most patients had chronicobstructive airways disease (COPD) without signs of reversibility. Ateach aerosol treatment, patients underwent monitoring of peak flowsbefore and after each treatment. Data for all patients is shown in FIG.11. Summary data of percent change in peak flow measurements is shown inFIG. 12. The average peak flow increased after aerosol interferon-γ,with significant increases in some patients. These data demonstrate thataerosol interferon-γ is safe and well tolerated in patients with chronicobstructive airways disease (COPD).

Example 14 Bronchoscopy

Bronchoscopy with bronchoalveolar lavage (BAL) was performed at baselineand after six months of treatment on the patient population of Example13 to evaluate the levels of certain pro-fibrotic and inflammatorycytokines. An optional bronchoscopy may be performed at the completionof treatment. Bronchoscopy with BAL was generally performed in a daysurgery setting. However, some subjects may be admitted overnight ifobservation is warranted. The procedure is performed as follows.

Each study patient was evaluated for bronchoscopy as per hospitalprotocol. Each evaluation includes measuring Hgb, platelets, BUN/CR,coagulation panel, ABG with PO₂≧65 mmHg, EKG, and chest x-ray.

Contraindications to bronchoscopy include lack of patient cooperation,recent myocardial infarction, malignant arrhythmias, refractoryhypoxemia, unstable bronchial asthma, partial tracheal obstruction orvocal cord paralysis, bleeding diathesis and uremia. Patients must beNPO at least 8 hours prior to bronchoscopy. An intravenous line wasplaced, supplemental oxygen was administered and continuous pulseoximetry and blood pressure monitoring was performed. Liquid and viscouslidocaine, a topical anesthetic, was used in the nasal passage andposterior pharynx for passage of the bronchoscope. During the proceduremidazolam and/or fentanyl may be administered to cause sedation anddecrease of the cough reflex. These medications are routinely used inbronchoscopy. The bronchoscope was passed through the nose and vocalcords, and an endobronchial exam was performed.

BAL was then performed in one or more segments of the lung byadministering 50 ml aliquots of sterile normal saline, for a total of upto 300 ml, and applying gentle suction for maximum return of fluid. TheBAL fluid was processed as follows. After BAL fluid was obtained fromthe patient, it was processed in the laboratory under the standardizedprotocol used for processing all BAL. The BAL fluid was filtered througha sterile gauze. A total cell count with differential was performed in ahemocytometer. Cell viability was determined by the Trypan Blue method.Ten cytocentrifuge slides were prepared from each lobe of BAL fluid andfrozen at −70° C. 24 hour supernatants were collected at a concentrationof 10⁶ cells/ml for cytokine ELISA assays. The volume of epitheliallining fluid was determined according to the protein method. Followingcentrifugation, BAL fluid supernatant was concentrated 10× to 50× usingthe AMICON filter method. Cytokine assays were performed with Luminexsystems (Minneapolis, Minn.). Luminex MAP, facilitates the simultaneousevaluation of multiple immune mediators with advantages of higherthroughput, smaller sample volume, and lower cost. It is a validalternative method to ELISA for the evaluation of the majority ofcytokines and for the characterization of immune system status.Cytokines were analyzed using the Luminex system includingInteferon-gamma IL-8 and TGF-beta. All samples were assayed intriplicate.

Each patient was monitored for potential side effects of bronchoscopy,including, but not limited to: fever, shortness of breath, hemoptysisand pneumothorax for 4 hours post procedure in the GCRC by the clinicalnursing staff.

Example 15

Ten IPF patients treated with INF-γ were followed for a period of 30weeks, nine IPF patients were followed for a period of 40 weeks, and sixIPF patients were followed for a period of fifty weeks. Thecharacteristics of the patients in the treatment group are provided inTable 3. Forced Vital Capacity (FVC) was measured by standard techniquesof pulmonary medicine. The results are demonstrated in FIG. 16. Thepatients as a group demonstrated an actual improvement in FVC in thesingle digit percentage range over the time monitored. This indicatesthat INF-γ may not only stabilize what is considered a graduallyprogressive condition but actually reverse some of the pulmonarydeterioration. IPF patients undergoing no treatment as represented bythe NYU Control Group of nine patients experienced a gradualdeterioration of pulmonary function exemplified by a reduction in FVCalso in the single digit percentage range over the same 30, 40 and 50week time period. The characteristics of the patients in the NYU ControlGroup are also provided in Table 3.

These INF-γ treatment and NYU Control groups were then compared tostudies performed by others using other treatments with IPF patients andproviding other control groups. The characteristics of the patients ineach group are also provided in Table 3. Again, a graphic display of thedeterioration in FVC over time is presented in FIG. 16. The patientsreceiving acetyl cysteine and the control group to which they arecompared are described by Demedts et al., N Engl J Med 2005; 353:21,hereby incorporated by reference. Patients receiving acetyl cysteine areidentified as acetyl cysteine Tx, and patients from the control group ofthis study are identified as acetyl cysteine control in FIG. 16. Thepatients receiving etanercept and the control group to which they arecompared are described by Raghu et al., Am J Resp Crit Care Med 2008;178: 948-955, hereby incorporated by reference. Patients receivingetanercept are identified as etanercept Tx, and patients from thecontrol group of this study are identified as etanercept placebo in FIG.16. Further, Intermune, Burlingame, Calif. conducted studies on IPFpatients using perfenadone as treatment. The results are generallyavailable from Intermune, Brisbane, Calif. and from internalcommunications at the company. In a first study involving 344 patientsreceiving 2403 mg perfenadone over 72 weeks, at the end there was a6.49% reduction in the FVC on average for patients receiving treatmentversus a 7.23% reduction in the FVC on average for patients receivingplacebo over the 72 week period. These data are presented in FIG. 16 asCapacity 1 PFD and Capacity 1 Placebo, respectively. In a second studyinvolving 435 patients receiving 2403 mg perfenadone over 72 weeks, atthe end there was a 6.49% reduction in the FVC on average for patientsreceiving treatment versus a 9.55% reduction in the FVC on average forpatients receiving placebo over the 72 week period. These data arepresented in FIG. 16 as Capacity 2 PFD and Capacity 2 Placebo,respectively.

The results provided in FIG. 16 demonstrate that patients suffering fromIPF experience not only arrest of further deterioration but actualimproved pulmonary function as demonstrated by increased FVC over time.To the contrary, patients receiving acetyl cysteine, etanercept,perfenadone or no treatment whatsoever demonstrate continueddeterioration of pulmonary function as evidenced by decreased FVC overtime.

TABLE 3 DEMOGRAPHICS Gender % FVC at % DLCO at Group N Mean Age M FBaseline Baseline INF Patients 10 69.3 ± 6.1 8 2 79.1 ± 13.4 47.7 ± 9.03NYU Placebo 9 71.1 ± 9.4 6 3 74.2 ± 19.3 49.4 ± 18.7 Acetylcysteine Tx80 62.0 ± 9.0 55 25 64.8 ± 15.4 43.0 ± 13.1 Acetylcysteine Placebo 7564.0 ± 9.0 60 15 66.6 ± 14.2 44.8 ± 15.2 Etanercept Tx 46 65.2 ± 7.7 3511 62.2 ± 11.9 36.3 ± 12.6 Etanercept Placebo 41 65.1 ± 7.1 24 17 61.1 ±12.7 36.9 ± 10.8 Intermune PFD Capacity 171 67 125 46 74.5 1 TxIntermune PFD Capacity 173 67 125 48 70.3 1 Placebo Intermune PFDCapacity 174 66 118 56 73 2 Tx Intermune PFD Capacity 174 67 129 45 73.62 Placebo Intermune PFD Capacity 174 67 129 45 73.6 2 Placebo

FIG. 13 provides the levels of interferon-γ measured in the BAL fluid ofsix of the ten patients both before and after treatment with aerosolizedinterferon-γ. FIG. 14 depicts the amount of TGF-β protein measured inBAL fluid from six IPF patients treated with aerosol interferon gamma.Pre—refers to baseline bronchoscopy and post is after a minimum of 24weeks of treatment. Units are picograms of TGF-β per ml of BAL fluid.FIG. 14 provides the levels of IL-8 measured in the BAL fluid of sixpatients both before and after treatment with aerosolized interferon-γ.

Example 16

Ten patients were administered 100 μg of recombinant INF-γ by oralnebulizer. The patients were then examined to determine the percentageof recombinant INF-γ that was deposited in various body compartments.Delivery to the lungs was assessed via radiolabeled gamma camerastudies. The results are provided in Table 4. The first column providesthe percentage of the amount of INF-γ that was deposited in the middlelobe of the lung compared to that deposited in the lung total. Thesecond column expresses the percentage of the entire dose that wasdeposited in the middle lobe of the lung. The third column expresses thepercentage of the entire dose that was deposited in the lung total. Thefourth column expresses the percentage of the entire dose that wasdeposited in the stomach. The fifth column expresses the exhaledactivity. The sixth column expresses the ratio of the amount of theentire dose that was deposited in the central lung compared to theamount of the entire dose that was deposited in the peripheral lung. Theseventh column expresses the ratio of the amount of the entire dose thatwas deposited in the central lung compared to the amount of the entiredose that was deposited in the peripheral lung in relation to how xenondeposits in the lung by ratio of central to peripheral deposition. Theeighth column expresses the percentage of the entire dose that remainedin the drug cup of the nebulizer.

TABLE 4 ML % % of Left Total % % in Lung ML % Lung % Stomach ExhaledaC/P sC/P drug Patient CTs Depo Deposition* Deposition* Activity*†deposition deposition cup MMAD 1 32.97 15.86 48.1 26.7 −5.4 1.16 1.31 24NA 2 25.89 14.01 54.1 10.1 2.4 0.85 1.10 21 1.9 3 28.70 18.65 65.0 2.011.6 0.64 0.88 16 1.8 4 27.31 17.59 64.4 2.5 −3.0 0.84 1.16 18 1.8 523.00 10.74 46.7 21.7 −2.0 0.82 1.17 16 2.0 6 29.00 15.74 54.3 1.1 0.30.83 1.22 27 2.1 7 33.3 23.7 5.4 0.78 1.13 28 1.3 8 48.0 6.0 7.3 0.751.21 26 1.3 9 26.3 17.02 64.7 6.5 −5.9 0.85 1.42 28 1.2 10  26.0 8.026.9 0.88 1.37 23 1.3 10  34.8 8.6 9.3 0.85 1.35 40 1.3 repeat *Dataexpressed as percent of nebulizable activity †Exhaled activity − Amountnebulized ‡ − (Total deposition by AC + I-neb base activity + mouthpieceactivity) ‡Amount nebulized = Initial nebulizer charge − post chamberactivity

1. A method for treating a pulmonary disease in a subject suffering froma pulmonary disease, comprising administering an aerosolized INF-γ in atherapeutically effective amount.
 2. The method of claim 1 wherein thepulmonary disease is idiopathic pulmonary fibrosis.
 3. The method ofclaim 1 wherein the pulmonary disease is mixed connective tissuedisease.
 4. The method of claim 1, wherein the pulmonary diseaseimproves so that a patient demonstrates an increase of at least 2% ofpredicted FVC relative to values from placebo.
 5. The method of claim 1,wherein INF-γ is administered in a dose and in a manner sufficient totransmit the INF-γ so that amounts of at least 100 picograms/milliliterof the INF-γ may be measured in the bronchoalveolar lavage (BAL) fluidof the subject.
 6. The method of claim 1, wherein INF-γ is administeredin a dose and in a manner sufficient to transmit the INF-γ so thatamounts of at least 150 picograms/milliliter of the INF-γ may bemeasured in the bronchoalveolar lavage (BAL) fluid of the subject. 7.The method of claim 1, wherein aerosolized INF-γ is administered at adose ranging from about 100 to 750 μg at least three times per week. 8.The method of claim 1, wherein aerosolized INF-γ is administered at adose of about 100 μg three times per week.
 9. The method of claim 1,wherein said administering results in deposition of INF-γ in the lungsof patients with pulmonary disease.
 10. The method of claim 1, whereinsaid administering results in deposition of about 40% of the INF-γ inlungs of patients with pulmonary disease.
 11. The method of claim 1,wherein said administering results in deposition of about 15% of theINF-γ in a middle lobe of lungs of patients with pulmonary disease. 12.The method of claim 1, wherein said administering results in depositionof about 60% of the INF-γ in lungs of patients with pulmonary disease.13. The method of claim 1, wherein INF-γ is administered in a dose andin a manner sufficient to provide a measurable decrease in the level ofIL-8 present in the bronchoalveolar lavage (BAL) fluid of a patient. 14.The method of claim 1, wherein INF-γ is administered in a dose and in amanner sufficient to provide at least a 10% decrease in the level ofIL-8 present in the bronchoalveolar lavage (BAL) fluid of a patient. 15.The method of claim 1, wherein INF-γ is administered in a dose and in amanner sufficient to provide at least a 20% decrease in the level ofIL-8 present in the bronchoalveolar lavage (BAL) fluid of a patient. 16.The method of claim 1, wherein INF-γ is administered in a dose and in amanner sufficient that the level of IL-8 in the BAL fluid of a patientsuffering from a pulmonary disease is reduced to an amount no more than50% more than the level of IL-8 in the BAL fluid of a normal controlsubstantially free of a pulmonary disease.
 17. The method of claim 1,wherein INF-γ is administered in a dose and in a manner sufficient toproduce at least a 10% increase in the level of TGF-β present in the BALfluid of a patient.
 18. The method of claim 1, wherein INF-γ isadministered in a dose and manner and with a nebulizer chosen so as toprovide delivery of at least about 60% of the INF-γ to the lungs of thesubject.
 19. A method of treating a subject having a pulmonary diseasecomprising delivering a therapeutically effective amount of anaerosolized INF-γ in combination with a therapeutically effective amountof an immunosuppressive or anti-inflammatory agent.
 20. The method ofclaim 19, wherein the immunosuppressive or anti-inflammatory agent isselected from the group consisting of a corticosteroid, azathioprine andcyclophosphamide.